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Disrupted homeostasis and ciliary defects in VPS13B deficient cells: implications for Cohen syndrome

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Universitat de Barcelona. Facultat de Farmàcia i Ciències de l'Alimentació

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[eng] Cohen syndrome (CS) is an ultra-rare, multi-system, autosomal recessive disorder caused by mutations in the VPS13B gene on chromosome 8q22.2. The most characteristic clinical features of this disorder are microcephaly, intellectual disability, progressive retinal dystrophy, hypotonia, intermittent neutropenia and truncal obesity.

Intriguingly, we noticed that CS patients share some clinical features with ciliopathy patients. Ciliopathies are a group of inherited disorders that are caused by defective cilia, small, antenna-like structures on the surface of the vast majority of cells that build tissues and organs in our body. These organelles assemble by an extension of the microtubule structure that constitutes the mother centriole, named axoneme. Since the axoneme protrudes beneath the plasma membrane, the ciliary membrane is formed as an extension of the plasma membrane. Yet, it has a distinct lipid and protein composition that is crucial to its function. Cilia are particularly important for embryo development and for the proper function of various organs. Patients affected by ciliopathies suffer from developmental delay, intellectual disability, obesity, skeletal abnormalities, vision and hearing loss, abnormal organ placement, kidney and liver cysts, and respiratory defects. Despite some overlap of clinical features in CS patients with those of established ciliopathies, VPS13B does not encode a ciliary protein and has not been linked to cilia assembly or function. Instead, VPS13B is associated with the Golgi and was suggested to have a role in maintaining Golgi ribbon structure, but its precise function remains unclear. Additionally, VPS13B belongs to the VPS13 protein family, a group of giant proteins suggested to act as lipid transporters at inter-organelle membrane contact sites, where they bridge membranes and form direct channels for lipid transport.

We speculated that VPS13B mutations may indirectly affect cilia by altering the lipid and protein composition of cilia, leading to some of the distinctive phenotypes in CS. To test this, I treated cultured cells with VPS13B siRNAs and analyzed cilia structure and composition. Apart from observing a wide range of cellular defects including mTORC1 downregulation, increased autophagy, G0 arrest and lysosomal structural defects, I also observed a variety of defects at the levels of the primary cilium. These include reduced levels of the lipid PI(4,5)P2 at the ciliary base, longer cilia and inability to activate the Hedgehog signaling pathway. These results suggest that VPS13B indirectly regulates ciliary membrane composition and function.

I also analyzed skin fibroblasts from CS patients and compared them with those of healthy controls. Interestingly, despite lacking the full length protein, patients still show a robust signal corresponding to VPS13B at the Golgi complex, suggesting that they might express alternative isoforms that still can localize to this organelle. However, CS cilia were very similar to controls. Curiously, treatment of CS fibroblasts with VPS13B siRNAs gives rise to a plethora of ciliary defects including Hedgehog inactivation. This supports the existence of shorter, at least partially functional VPS13B isoforms in CS patients. To understand if this situation was general or specific to the skin, I decided to investigate disease-relevant tissues. I derived neural rosettes from induced pluripotent stem cells of a healthy control and a CS patient. Interestingly, neural progenitor cells (NPCs) from CS rosettes presented also longer and abnormal cilia compared to control NPCs, suggesting the occurrence of a ciliary impairment in brain-related tissues.

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Tesis Doctorals - Facultat - Farmàcia i Ciències de l'Alimentació

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Disrupted homeostasis and ciliary defects in VPS13B deficient cells: implications for Cohen syndrome