Iaconis, Daniela (2012) The Oral-facial-digital type I syndrome: a model to study renal cystic disease in ciliopathies. [Tesi di dottorato] (Inedito)

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Abstract

The Oral-Facial-Digital type I syndrome (OFDI; MIM 311200) is a rare syndromic form of inherited renal cystic disease. It is transmitted as an X-linked dominant, male lethal disorder and is caused by mutations in the OFD1 gene. Previous studies demonstrated that OFDI belongs to the growing number of disorders ascribed to dysfunction of primary cilia. Among the different clinical signs OFD type I is characterized by the presence of renal cystic disease, which is present in over 60% of cases. With the purpose of studying the role of the Ofd1 transcript in renal cystic disease, we generated two conditional Ofd1 deficient mouse models (Ksp and IND), which resulted in viable mice characterized by renal cystic disease and progressive impairment of renal function. The study of these models allowed us to demonstrate that primary cilia initially form and then disappear after the development of cysts, suggesting that the dysfunction of primary cilia is a consequence rather than the primary cause of renal cystic disease. Immunofluorescence and western blotting analysis revealed upregulation of the mTOR pathway in both dilated and non dilated renal structures. Treatment with rapamycin, a specific inhibitor of the mTOR pathway, resulted in a significant reduction in the number and size of renal cysts and a decrease in the cystic index compared with untreated mutant animals, suggesting that cystogenesis in our model is mTOR-dependent. mTOR is frequently deregulated in inherited forms of cystic kidney although the molecular mechanisms underlying this phenomenon are still to be determined. The use of both in vitro and in vivo systems allowed us to demonstrate that OFD1 has a role in the formation of the pre-initiation complex of translation (PIC). We showed that OFD1 coimmunoprecipitates with two subunits ( and ) of the eIF3 complex and colocalizes at the centrosome with subunits of the PIC. In addition, we also demonstrated that OFD1 is present in polysomes extracted from kidneys and interacts in vivo with subunits of the eIF3 complex. Microarray experiments performed on mRNA extracted from polysomes indicate impaired translation for a number of transcripts. These results indicate for the first time a role of OFD1 in the formation of the PIC and in the regulation of translation. Among the putative targets we focused our attention on GH and our data suggest that higher levels of the GH due to the impairment of translation may be responsible for the upregulation of the mTOR pathway. All together our results shed light on the pathogenetic mechanisms underlying this rare inherited form of renal cystic disease and propose new possible link between renal cystic disease and deregulation of the mTOR pathway.

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