Biology subjects

Takla, T. N.

Publications and source records attributed to Takla, T. N..

3 recordsLinked to original sources

bioRxiv2023

Genome-wide CRISPRi Screen in Human iNeurons to Identify Novel Focal Cortical Dysplasia Genes

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels. Interestingly, positive hits were enriched for brain-specific genes, highlighting the effectiveness of using human iPSC-derived induced neurons (iNeurons) in our screen. We investigated the signaling pathways of six candidate genes: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. All six genes increased phosphorylation of S6. However, only two genes, PIK3R3 and HIP1, caused hyperphosphorylation more proximally in the AKT/mTOR/S6 signaling pathway. Importantly, these two genes have recently been found independently to be mutated in resected brain tissue from FCD patients, supporting the predictive validity of our screen. Knocking down each of the other four genes (LRRC4, EIF3A, TSN, and URI1) in iNeurons caused them to become resistant to the loss of growth factor signaling; without growth factor stimulation, pS6 levels were comparable to growth factor stimulated controls. Our data markedly expand the set of genes that are likely to regulate mTOR pathway signaling in neurons and provide additional targets for identifying somatic gene variants that cause FCD. Significance StatementFocal cortical dysplasia (FCD) is a common cause of focal epilepsy due to somatic variants in mTOR pathway genes in FCD brain tissue. Unbiased sequencing to identify novel FCD genes is challenging since these variants are often in a small subset of neurons. To overcome this challenge, we used an in vitro genetic screen in human neurons using an FCD biomarker, uncovering genes that increase neuronal mTOR signaling when their expression is lost. Two candidate genes were mutated in patient FCD brain tissues in a recent study, supporting a causative role for these genes in FCD. Our work suggests gene candidates for somatic variant analysis in FCD tissue and indicates added value for genetic screening in neurons over cell lines.

neuroscience↗

bioRxiv2023

A shared pathogenic mechanism for valproic acid and SHROOM3 knockout in a brain organoid model of neural tube defects

Neural tube defects (NTDs) including anencephaly and spina bifida are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or life-long severe complications (spina bifida). Despite hundreds of genetic mouse models having neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals such as antiseizure medications have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette spheroid (SOSRS) brain organoid system, we have developed a high-throughput image analysis pipeline for evaluating SOSRS structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. This expansion was mimicked by GSK3{beta} and HDAC inhibitors; however, RNA sequencing suggests VPA does not inhibit GSK3{beta} at these concentrations. Knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity.

neuroscience↗

bioRxiv2022

Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies. SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.

neuroscience↗