Amplification of human interneuron progenitors promotes brain tumors and neurological defects

Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development. Description Physiopathology in the human brain Development of the human brain involves processes not seen in most animals. Eichmüller et al. identified CLIP cells, a human progenitor cell type responsible for tuberous sclerosis, a rare form of epilepsy triggered by excessive mechanistic target of rapamycin (mTOR) signaling and recapitulated the disease in human cerebral organoids (see the Perspective by Ihrie and Henske). Excessive CLIP cell proliferation causes tumorigenesis and brain abnormalities. The researchers found that the sensitivity of these cells for mTOR signaling, not loss of healthy alleles, is responsible for the disease. This work changes our understanding of neurodevelopmental disease and reveals crucial aspects of human brain development. —BAP Cerebral organoids provide mechanistic insights into human-specific brain development and the disorder tuberous sclerosis. INTRODUCTION Development of the human brain is protracted and involves distinct processes that have contributed to its large size and complexity. However, these changes might have also increased the vulnerability of the human brain to genetic disorders. Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder characterized by debilitating neuropsychiatric symptoms, including epilepsy, autism, and intellectual disabilities. It involves distinct morphological aberrations in the brain such as the formation of benign subependymal tumors and dysplastic cortical lesions, which are commonly referred to as cortical tubers. TSC arises from mutations in the mechanistic target of rapamycin (mTOR) inhibitors TSC1 and TSC2 and is thought to be caused by elevated mTOR signaling activity. RATIONALE TSC is thought to originate from heterozygous TSC1/2 germline mutations followed by loss-of-function mutations that disrupt the second allele and cause somatic loss of heterozygosity (LOH). This hypothesis is generally supported by mouse models, but analysis of patient tissues revealed LOH only in tumors and rarely in the dysplastic regions. In addition, the animal models failed to recapitulate the full spectrum of pathognomonic lesions. We hypothesized that aspects of specifically human brain development rather than LOH could initiate the disease. RESULTS We generated a human model for TSC by growing cerebral organoids from patients who carried mutations in TSC2. The organoid model recapitulates the emergence of both brain tumors and dysplastic cortical regions. Using single-cell RNA-sequencing (scRNA-seq) and extensive histological validation, we identified a specific interneuron progenitor population that gives rise to both tumor and cortical tuber lesions. Comparisons of expression signatures with fetal brain scRNA-seq data revealed the origin of this cell type in the caudal ganglionic eminence (CGE) during mid-gestation. We therefore refer to this cell type as caudal late interneuron progenitor (CLIP) cells. Our analysis uncovered particularly low amounts of TSC1/2 complex proteins in CLIP cells, making them susceptible to TSC1/2 levels. CLIP cells overproliferate and initiate both tumor and cortical tuber formation even when just one copy of TSC2 is lost. The second TSC2 allele can be mutated during tumor progression, but this does not occur through a second somatic mutation. Instead, it is caused by copy-neutral LOH (cnLOH), the exchange of large genomic regions between homologous chromosomes. CLIP cells depend on epidermal growth factor receptor (EGFR) signaling, and EGFR inhibition can revert the TSC phenotype, suggesting an alternative inroad to therapeutic intervention. The sequence of events leading to TSC was validated in postmortem patient tissues. The seemingly confusing histological appearance of TSC brain lesions can be explained by the initiation of cortical lesions by CLIP cells and their derivatives, whereas other cell types contribute only at later stages. Thus, our analysis can explain the different mutational status of brain tumors and cortical tubers in TSC patients in the context of a common cell of origin. CONCLUSION This work shows that the analysis of neurodevelopmental genetic disorders can lead to fundamental mechanistic insights into human brain development. CLIP cells are identified as the shared cell of origin for brain lesions in TSC. The contribution of different lineages over time then generates the complex neurological defects observed in TSC. Although cnLOH occurs during TSC tumor progression, it is dispensable for disease initiation, demonstrating that the disease relevance of mutations should always be evaluated in the context of a specific cell of origin. Human brain expansion was accompanied by a diversification of progenitor cell types. This work showcases an example of such a human progenitor cell type responsible for a human neurodevelopmental disease and demonstrates the necessity of using human models to identify disease mechanisms that involve processes not conserved in all mammals. During mid-gestation, CLIP cells residing in the CGE generate interneurons that migrate into the cortex. (Top right) In TSC, CLIP cells generate brain tumors and cortical tubers. Heterozygous mutations in TSC2 result in excessive proliferation of CLIP cells, generating cell types of cortical tubers (orange) as well as brain tumors (red). During progression, the healthy allele is lost because of cnLOH, increasing tumor proliferation. IMAGE: KELLIE HOLOSKI/SCIENCE