Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: Exploring new functions of the Nijmegen Breakage Syndrome gene in cerebellar development and tumorigenesis

Medulloblastoma (MB) is the most common pediatric tumor that stems from the transformation of granule cell progenitors (GCPs) in the developing cerebellum. Mutations in DNA Damage Response (DDR) genes have been associated with MB, indicating a role for DDR proteins in tumor insurgence. Hypomorphic mutations of the NBN gene, whose encoded protein NBS1 is essential for the orchestration of complex cellular responses to replication stress and DNA ruptures, causes the DDR-defective Nijmegen Breakage Syndrome (NBS), a rare human autosomic recessive disorder. The clinical manifestations of NBS have been predominantly ascribed to the defective activation of DNA repair mechanisms. However, the extreme vulnerability of the Nervous System (NS), reflected in a strongly microcephalic phenotype which is a common feature to a diverse range of DDR-defective disorders, is still poorly understood. The neurological features of NBS patients have been recapitulated in a central nervous system (CNS)-restricted Nbn-KO animal model, which manifests phenotypes such as microcephaly, ataxia and cerebellar hypoplasia. Strikingly, mice with CNS-conditional KO of the SHH pathway, the main driver of GCP expansion during cerebellar histogenesis, show similar phenotypes, suggesting a possible overlap between NBS1 and the SHH pathway. We have recently demonstrated that CNS-specific ablation of Nbn completely abrogates the formation of SHH-dependent tumorigenesis and, furthermore, causes severe cerebellar defects during post-natal development. Of note, derangement of the cerebellar architecture is associated with a strong impairment of the SHH pathway, suggesting an epistasis of NBS1 function on the SHH pathway. Given that: i) there is an emergent link between DDR and centrosome proteins; ii) NBS1 localizes at the centrosomes; iii) during interphase the centrosome converts into the basal body, which enucleates the Primary Cilium (PC); iv) the PC is essential for SHH signaling; v) MRE11 mutations are involved in ciliopathies; we provocatively raised the hypothesis that NBS1 regulates the SHH pathway through a new uncanonical role on primary ciliogenesis, which subsequently impacts on the development of the cerebellar cortical architecture and on GCP tumorigenic transformation. In this study, through the generation and in-depth characterization of a new mouse model defined by a GCP-restricted contextual inactivation of NBS1 and Ptch1, we not only demonstrate that NBS1 loss in GCPs abrogates Ptch1-dependent MB formation, but it is also responsible for an aberrant development of the cerebellar tissue coupled with a significant impairment of the SHH pathway in a cell autonomous framework. Accordingly, by exploiting mice with the sole deletion of Nbn, we show that its GCP- restricted KO is sufficient to induce an aberrant cerebellar cortical patterning; this is evinced by a reduced cerebellar size with impaired proliferation and differentiation of GCPs, coupled with a severe impairment in the activity of the SHH pathway. Of interest, we show that NBS1-deficient cells consistently display severe alterations in their ciliary structure in an in vivo, ex vivo and in vitro context. Furthermore, NBS1 does not only localize at the centrosome, but also at the base of the PC (basal body) in both physiological and Replication Stress (RS)-induced backgrounds, further strengthening the hypothesis of its new function on PC regulation. Collectively, our results uncover a possible novel role of NBS1, in addition to its recognized one in DDR, in regulating ciliary morphology. Through this novel function it could influence the activity of the SHH pathway and consequently impact on SHH-driven cerebellar development/tumorigenesis, alongside offering an alternative reading frame for the yet inexplicable susceptibility of the NS to defects in the DDR.