MODELING HOUGE‑JANSSENS SYNDROME TYPE 1 IN ZEBRAFISH TO INVESTIGATE THE ROLE OF PPP2R5D‑LINKED BEHAVIORAL DEFICITS AND PARKINSONISM

Houge-Janssens syndrome (HJS) type 1, arising from PPP2R5D mutations, causes intellectual disability, macrocephaly, autism and epilepsy. Single‑point mutations such as E198K/E200K are associated with parkinsonism, linking neurodevelopmental processes to cognitive and motor dysfunctions. However, the cellular mechanisms underlying PPP2R5D pathophysiology remain poorly understood. We use zebrafish to bridge mutations to behavioral and cellular mechanisms as it provides a powerful model (small, genetically and optically accessible, high fecundity) enabling high‑throughput comparisons of mutation effects across hundreds of siblings. First, we used RT-qPCR to confirm ppp2r5d expression during early developmental stages (0-6dpf) on whole larvae and in adult brain and spinal cord. To map expression of ppp2r5d during development, we performed HCR experiments confirming ubiquitous expression in the brain and spinal cord. To understand the role of ppp2r5d, we generated knock-out fish mutant (93% somatic mutation rate) by using CRISPR–Cas9. To model HJS type 1, we developed a tol-mediated transgenesis to overexpress WT versus E198/200K human PPP2R5D. To monitor macrocephaly phenotype, we will measure brain size across genetic conditions to test that may correlate with deficits in brain development. To screen for behavioral defects in ppp2r5d knock-out and overexpression mutants, we developed a mid-throughput high frequency behavioral setup assessing sensorimotor integration, habituation and kinematics of locomotion and inspired by human studies on cognitive disorders combining light/dark assay with roving oddball paradigm. Future work will involve the generation of a knock-in of the E198/200K nucleotide and perform the same pipeline to probe cellular specificity and circuitry to explain emerging phenotypes.