Periodic Reporting for period 1 - CODICES (Exploring new genetic causes and pathological mechanisms of epileptic focal cortical dysplasia)
Periodo di rendicontazione: 2021-09-01 al 2023-08-31
Focal cortical dysplasia (FCD) is a rare, genetic, non-syndromic developmental malformation of the cerebral cortex that accounts for 5-10% of patients with focal epilepsy, and that represents the main cause of pharmaco-resistant epilepsy. FCD affects mainly children and young adults, and nowadays the only treatment available is invasive surgical resection of the brain epileptogenic zone, which is effective in only 62% of cases. Thus, a better understanding of the disorder is urgent in order to point towards strategies that would allow to generate new, more effective and less invasive treatments.
Among FCD subtypes, FCD type 2 (FCD2) is characterized at the histopathological level by an abnormal structure of the cortex in the area affected by the disorder, where the characteristic six-layer structure appears disrupted. Abnormally large (cytomegalic) cells are also a classic hallmark of FCD2. Around 60% of FCD2 cases are due to inherited germline and/or de novo somatic mutations in genes regulating the mechanistic target of rapamycin (mTOR) pathway, which regulates cell growth, metabolism and proliferation. These mutations always cause a hyperactivation of the mTOR pathway that is thought to be associated with the aforementioned hallmarks of the disorder. Together with its focal nature, the role of somatic mutations in causing the disorder has recently defined FCD as a mosaic disorder of the brain. Despite these findings, a clear understanding of the consequences of mTOR pathway mutations, and especially mosaic mutations, in the development of the cerebral cortex and the insurgence of the disorder is still missing.
This project addressed the genetic, cellular and molecular mechanisms underlying the pathophysiology of FCD2 by modeling the disorder in vitro using 3-dimensional culture systems called cortical organoids that are generated from patient-derived human induced pluripotent stem cells (hiPSCs). This system has the advantage of reproducing developmental features specific to humans, and thus overcomes existing limitations encountered in mouse models, where the disorder is only partially reproducible.
Among FCD subtypes, FCD type 2 (FCD2) is characterized at the histopathological level by an abnormal structure of the cortex in the area affected by the disorder, where the characteristic six-layer structure appears disrupted. Abnormally large (cytomegalic) cells are also a classic hallmark of FCD2. Around 60% of FCD2 cases are due to inherited germline and/or de novo somatic mutations in genes regulating the mechanistic target of rapamycin (mTOR) pathway, which regulates cell growth, metabolism and proliferation. These mutations always cause a hyperactivation of the mTOR pathway that is thought to be associated with the aforementioned hallmarks of the disorder. Together with its focal nature, the role of somatic mutations in causing the disorder has recently defined FCD as a mosaic disorder of the brain. Despite these findings, a clear understanding of the consequences of mTOR pathway mutations, and especially mosaic mutations, in the development of the cerebral cortex and the insurgence of the disorder is still missing.
This project addressed the genetic, cellular and molecular mechanisms underlying the pathophysiology of FCD2 by modeling the disorder in vitro using 3-dimensional culture systems called cortical organoids that are generated from patient-derived human induced pluripotent stem cells (hiPSCs). This system has the advantage of reproducing developmental features specific to humans, and thus overcomes existing limitations encountered in mouse models, where the disorder is only partially reproducible.
We generated cortical organoids starting from hiPSCs that we derived from patients affected by FCD2, and carrying mutations in mTOR pathway genes. In order to address the role of genetic mosaicism in the developmental insurgence of FCD2, we generated mosaic organoids by mixing mutant and control hiPSCs. Organoids were grown for a maximum of 6 months in culture and harvested at different timepoints to address 1) the appearance of FCD2 features such as mTOR hyperactivity and presence cytomegalic abnormal cells, and 2) the cellular and molecular mechanisms perturbed by the mutation. Towards these objectives, we applied technologies such as immunostaining and (confocal) microscopy, and single-cell transcriptomics. Our model successfully reproduced some of the characteristic features of FCD such as mTOR hyperactivity and abnormal cells. Furthermore, our data highlight perturbed progenitors and cell differentiation in the cortex due to mTOR hyperactivity.
Thus, our results will provide a clearer view especially of the developmental cellular and molecular mechanisms that are perturbed due to FCD2 mutations, and the consequences of pathogenic mosaicism for the development of the cortex. Our study will clarify the insurgence of this rare developmental mosaic disorder of the brain, while at the same time providing a first human-specific mosaic model of the disorder. This methodology will be exploited to prompt future studies that will address other epilepsy-related neurodevelopmental genes, but will also serve as a platform for the development of more targeted pharmacological treatments.
The results of this study will be published in a gold open access manuscript that is now in preparation, and will be presented to the scientific community in the framework of dedicated neuroscience, genetics and epilepsy conferences that are taking place in 2024.
Thus, our results will provide a clearer view especially of the developmental cellular and molecular mechanisms that are perturbed due to FCD2 mutations, and the consequences of pathogenic mosaicism for the development of the cortex. Our study will clarify the insurgence of this rare developmental mosaic disorder of the brain, while at the same time providing a first human-specific mosaic model of the disorder. This methodology will be exploited to prompt future studies that will address other epilepsy-related neurodevelopmental genes, but will also serve as a platform for the development of more targeted pharmacological treatments.
The results of this study will be published in a gold open access manuscript that is now in preparation, and will be presented to the scientific community in the framework of dedicated neuroscience, genetics and epilepsy conferences that are taking place in 2024.
In Europe, around 100,000 children and adolescents are diagnosed with epilepsy every year, and FCD represents the main cause of epilepsy that is not treatable pharmacologically but instead requires invasive surgery. Recent progress in genetics has proven the role of somatic mosaicism in the insurgence of these disorders thus, providing a genetic diagnosis to many families. However, since these genetic discoveries are quite recent, little progress has since been made in modeling the disorder to explore its mechanisms of insurgence. Furthermore, rodent models generated so far have highlighted the need to study the disorder in a context that is capable of reproducing human-specific neurodevelopmental features. Our project fills these needs by providing a first human-specific mosaic model of FCD using cortical organoids generated from patient-derived hiPSCs. Our results provide a better understanding of the cellular and molecular mechanisms impacting cortical development and explaining the disorder insurgence. This will have a significant impact in the field by elucidating fundamental aspects that were so far unexplored, in order to explain a rare and severe disorder of the brain that represents a major burden for affected patients and their families. We hope that our study will prompt future work towards the generation of effective pharmacological treatments that could replace invasive surgery.