PhD Student University of Manitoba, Manitoba, Canada
Abstract: Rett syndrome (RTT) is a rare neurodevelopmental disorder with limited treatment options. RTT is caused by mutations in the MECP2 gene, a key regulator of brain development. Individuals affected by RTT exhibit variable severity in clinical presentation, which cannot be completely explained by mutation type alone. Therefore, we consider genetic modifiers, as they can alter disease severity and may inform therapeutic targets. A previous RTT modifier screen in Mecp2/Y mice found that deleterious mutations in the DNA repair gene Fan1 increased longevity and improved health in the animal model. To determine the translational relevance to the primarily female RTT population, we aim to examine if FAN1 ablation alters 2D and 3D RTT human induced pluripotent stem cell (hiPSCs) derived models for molecular phenotypes relevant to RTT disease biology. An isogenic RTT hiPSC line was edited to insert a stop codon in exon 1 of FAN1, resulting in an RTT FAN1KO line. The RTT FAN1KO, RTT and isogenic control hiPSCs were differentiated into neural precursor cells (NPCs) and bulk RNA sequencing was performed. Ablation of FAN1 resulted in over 50 DEGs between RTT and RTT FAN1KO NPCs. Gene set enrichment analysis found that ablation of FAN1 in RTT NPCs resulted in the upregulation of neuronal processes such as “regulation of neurotransmitter levels”, “positive regulation of synaptic transmission,” and “regulation of dendritic extension,” suggesting that FAN1 ablation may alter aberrant neuronal morphology and activity in RTT. At the single-cell transcriptional level, RTT dorsal forebrain organoids (DFOs) indicate aberrant neuronal processes. Initial single-nuclei RNA sequencing (snRNA-seq) data of RTT DFOs show downregulation of neuronal processes such as “regulation of post-synapse organization and regulation of neuron migration” in RTT inhibitory neurons compared to control. Further analysis with the deep learning model NEUROeSTIMater found that RTT inhibitory and corticofugal projection neurons showed increased neural activity compared to control. Further functional experiments will be performed to investigate the impact of FAN1 ablation on neuronal activity and morphology in RTT DFOs. These transcriptional level analyses have revealed aberrant neuronal processes in RTT that may be ameliorated via FAN1 ablation.
Funding Source: This project was supported by Brain Canada, CIHR, NSERC, Children's Hospital Research Institute of Manitoba, Research Manitoba, the Ontario Rett Syndrome Association and NSERC Canada Research Chair funding.
