Development of in vitro model of Joubert Syndrome to individuate possible pathogenetic mechanisms of the disease.

Joubert Syndrome (JS) is a rare neurodevelopmental disease belonging to the group of ciliopathies, characterized by defects on the cerebellar vermis and malformation of the brain stem. These structural anomalies are linked to neurological dysfunctions such as hypotonia, developmental delay, motor impairment, and abnormal breathing. All known pathogenic variants in JS affect genes encoding proteins crucial for the primary cilium (PC) — a microtubule-based organelle involved in cellular signaling, development, and homeostasis; however, very little is known about the role of primary cilia in the onset of Joubert Syndrome. Jouberin is a little-known protein linked to Joubert Syndrome (JS), however, its role and mechanism in the disease remain unclear. The purpose of this project is to investigate the impact of Jouberin mutations on cilium structure and function, with a focus on their consequences on neural development and maturation. The early onset of JS during neurogenesis makes investigating these mechanisms highly complex. To overcome this and investigate the impact of Jouberin on PC and, consequently, on neurodevelopment, the use of Neural Stem Cells derived from patient-iPSCs represents a strong model for disease characterization. We began this thesis by analyzing fibroblasts derived from JS patients carrying AHI1 mutations. These primary cells were used to investigate how the mutations affect Jouberin function, as well as primary cilium (PC) assembly, disassembly, and morphology, and their possible connection to cell cycle regulation. Subsequently, the fibroblasts were reprogrammed into human induced Pluripotent Stem Sells (hiPSCs) and differentiated into Neural Stem Cells (hiNSCs). The resulting JS-derived neurospheres represent a valuable model that faithfully reproduces the defective NSC phenotypes and the abnormalities in neural differentiation and maturation observed in JS patients. Through these cellular models — fibroblasts, hiPSCs, and their neural derivatives — we aim to define the AHI1-related ciliopathy phenotypes in detail and uncover novel insights into the cellular and molecular pathogenesis of JS. This integrated platform also offers a foundation for identifying new disease mechanisms and guiding future therapeutic strategies.