Cascone, Emanuela (2025) Hepatic organoids: advancing Wilson’s disease research. [Tesi di dottorato]

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Abstract

Wilson's disease is a rare genetic disorder caused by mutations in the ATP7B gene, which encodes a copper-transporting ATPase essential for hepatic copper homeostasis. Under normal conditions, ATP7B localizes to the Golgi complex in hepatocytes but translocates to the endo-lysosomal compartment in response to copper overload to facilitate copper excretion via bile canaliculi. The H1069Q mutation, the most common in Caucasian populations, disrupts ATP7B folding, leading to its retention and degradation in the endoplasmic reticulum (ER), preventing its proper localization and resulting in pathological copper accumulation. Currently, the study of the H1069Q mutation in Wilson's disease relies on isogenic hepatocyte cultures in 2D systems. While these models have provided valuable insights, their two-dimensional nature fails to replicate the complex cellular interactions and architecture of the liver, limiting their ability to fully represent the in vivo pathophysiology of the disease. To address these limitations, the present study introduces a novel advanced model for investigating Wilson's disease: hepatic organoids (HOs). This model provides a more physiologically relevant representation of the disease by enabling the study of both parenchymal and non-parenchymal liver cells, thus capturing a comprehensive disease phenotype. Using different approaches, including qPCR, western blot, and immunofluorescence, I demonstrated that the H1069Q mutation induces a hepatic phenotype characterized by ductular reaction and fibrosis. The effect of copper overload on this mutation was further explored through RNA-seq, revealing a significant activation of the PERK-ATF4-CHOP pathway, an ER-stress-induced pathway. Finally, the potential therapeutic effects of Domperidone, an HSP70 inhibitor, were evaluated, focusing on the PERK pathway. Treatment with Domperidone appeared to mitigate the ER-stress response to copper overload. These findings underscore the utility of hepatic organoids as a powerful model for studying the molecular mechanisms of Wilson's disease and for screening potential therapeutic candidates, advancing efforts to develop targeted treatments.

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