Abstract
Jordan’s Syndrome is rare, genetically-linked neurodevelopmental disorder caused by variants in Protein Phosphatase 2 Regulatory Subunit B’ Delta (PPP2R5D) and is characterized by intellectual disability (ID), delayed motor skill and speech development, and distinct facial features such as a prominent forehead, long face, and widely-spaced eyes. Jordan’s Syndrome is caused by missense mutations that result in a dominant-negative genotype; one defective copy of the PPP2R5D gene in each cell can affect development. PPP2R5D encodes a regulatory subunit of a major protein phosphatase family called PP2A and is thought to be involved in the proper development of neurons and the signaling between them. Advancements in disease modeling provide a potential starting point of investigation to elucidate the molecular underpinnings of PPP2R5D-related ID. Specifically, induced pluripotent stem cells (iPSCs) have become a valuable tool to model human disease containing mutations and the genetic background from diagnosed individuals. Here, I investigated how one of the most common variants of PPP2R5D, E198K, influences the molecular, cellular, and developmental function of the protein. The aim for this specific research project was to establish both cellular and molecular phenotypes of the E198K variant patient-derived iPSCs, iPSC-derived neural stem cells (NSCs), and iPSC-derived neurons to compare gene expression, protein expression, and cell morphology to isogenic controls containing an identical genetic background with the variant corrected. Patient-derived fibroblast cells were acquired and reprogrammed to iPSCs. iPSCs were then differentiated into NSCs and neurons and characterization was performed on all cell types at defined stages during the differentiation process. To evaluate gene expression, we used reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and our results indicate markers characteristic of iPSCs, NSCs, and were either significantly up- or downregulated as a result of the E198K variant. RT-qPCR analysis also showed that genes comprising the PP2A holoenzyme and additional genes comprising the regulatory subunit of PP2A were altered in patient-derived fibroblasts, and expression levels of the AKT-mTOR signaling cascade were also altered as a consequence of the variant. To semi-quantify protein expression, we used western blotting and probed for target proteins involved in the AKT-mTOR pathway. Our results indicated no significant difference in expression levels between the E198K variant and isogenic control upon analysis of each individual cell type, but when overall protein expression was assessed across all cell types combined, we found that phosphorylated glycogen synthase kinase 3 beta (P-GSK3ß) was significantly higher in the E198K variant compared to the isogenic controls. To confirm the differentiation stage of each cell type, immunocytochemistry and fluorescence microscopy were used. Detection of positive signal was indicative of successful staining as compared to negative controls. Characterizing these various cell types in vitro was a critical first step in exploring potential therapeutic platforms for individuals diagnosed with this rare, genetically-linked neurodevelopmental disorder and may provide insight into the pathophysiology of other IDs as well.