Titolo della tesi: A new role of NBS1 in primary ciliogenesis: implications for the Nijmegen Breakage Syndrome
The maintenance of genomic integrity is strictly dependent on the DNA damage response (DDR). Defects in key DDR genes lead to human disorders known as DDR-defective syndromes, characterized by predisposition to cancer, immunodeficiency, and developmental defects, especially in neural tissues. NBS1, together with MRE11 and RAD50, is part of the MRN complex, a crucial sensor of DNA double-strand breaks (DSBs), as well as a primary actor in DNA repair and DDR activation. In humans, hypomorphic mutations in each component of the complex cause DDR-defective syndromes characterized, among other features, by microcephaly. In particular, mutant forms of NBS1 are responsible for a rare human autosomic recessive disorder, the Nijmegen Breakage Syndrome (NBS). So far, the neurological phenotypes of these syndromes were interpreted as a consequence of the defective activation of DNA repair mechanisms. However, this is not sufficient to fully explain the extreme vulnerability of the central nervous system (CNS) to DDR defects.
Of interest, microcephaly is a common outcome of mutations in proteins operating at the centrosome or at the primary cilium (PC). This is an antenna-like organelle essential for the regulation of specific neurodevelopmental signaling pathways like the Sonic Hedgehog (SHH) pathway, which drives the physiological expansion of cerebellar granule cell progenitors (GCPs). Moreover, various DDR proteins, including the members of the MRN complex, localize at the centrosome. We have recently demonstrated that CNS-restricted NBS1 KO completely abrogates cerebellar development and tumorigenesis, and correlates with a strong impairment of the SHH pathway both in vivo and ex vivo, suggesting an epistatic function of NBS1 on SHH pathway. In this work, we further confirmed the SHH pathway downregulation by NBS1 depletion in different cell-autonomous murine models.
On the base of all these data we raised the hypothesis that NBS1/MRN complex may regulate the SHH pathway through a new uncanonical role on primary ciliogenesis, with consequences on development of the cerebellar cortical architecture.
Accordingly, we provided strong evidence that loss of NBS1 determines severe defects in ciliogenesis in different primary or immortalized murine and human cell models. We observed that all the components of the MRN complex localize at the Basal Body (BB) of the PC. However, while MRE11 depletion or pharmacological inhibition by mirin associates with PC elongation and alterations of its morphology, the absence of RAD50 does not affect primary ciliogenesis. Furthermore, different DNA-damage inducing drugs alter neither PC length nor NBS1 localization to the centrosome/BB, suggesting that NBS1 could regulate primary ciliogenesis independently of the DDR. Finally, we showed that HFs derived from NBS patients consistently display alterations in their ciliary structure, despite the mutated NBS1 still localizes at the centrosome/BB.
Collectively, our results uncover a possible novel role of NBS1/MRN complex in regulating primary ciliogenesis. Through this novel function, NBS1 could influence the SHH pathway and consequently impact on cerebellar development, which would provide a new interpretative perspective for the neurodevelopmental defects observed in NBS patients.