Cerebral Cortical-Like Organoid Model of PPP2R5D Induced Genetic Intellectual Disability Displays Variant-Specific Disease Severity Phenotype

Jordan’s Syndrome (JS) is a rare, neurodevelopmental disorder caused by de novo missense mutations in protein phosphatase 2 regulatory subunit B’delta ( PPP2R5D ). JS is characterized by severe neurological impairments starting in early life. PPP2R5D encodes for B56δ, one of the regulatory subunits of protein phosphatase 2A (PP2A). PP2A is a heterotrimeric protein serine/threonine phosphatase that is highly expressed in the brain and the liver. Past studies have focused on PP2A’s role in liver and little is known about the holoenzyme’s behavior in neuronal cells. Although B56δ is known to play an important role in the substrate specificity of PP2A, the identification of validated downstream substrates in JS remains unclear. To better understand how the mutations affect neuronal cells, we developed cerebral cortical-like organoids from an engineered allele series of the most common JS mutations to characterize the physiological changes throughout different stages of neurodevelopment. Organoids were assessed for transcriptomic, protein, and electrophysiological changes utilizing bulk RNA sequencing, immunocytochemistry, Western Blot, and high-density MicroElectrode Array. The results identify differentially expressed genes and translated proteins, potential neuronal substrates, and significant electrophysiological signatures that suggest mutations in B56δ lead to variant-specific dysfunction of PP2A. Overexpression of PPP2R5D through AAV transduction of organoids rescued several phenotypes in the variants, suggesting different pathogenetic etiology underneath. Our findings successfully characterized cerebral cortical-like organoids in JS cell lines and demonstrated its potential as a model for studying neurodevelopmental disorder and for screening therapeutic approaches.