Thesis title: NBS1 regulates primary ciliogenesis altering actin/tubulin dynamics: implications for the Nijmegen Breakage Syndrome.
Genome integrity must be preserved for the correct propagation of the genetic information. To safeguard this, cells have evolved repair pathways, collectively known as the DNA Damage Response (DDR). Mutations in NBS1, a subunit of the MRE11/RAD50/NBS1 (MRN) complex, lead to the DDR-defective Nijmegen Breakage Syndrome (NBS), a rare human autosomal recessive disorder. The clinical manifestations of NBS are microcephaly, neurological abnormalities, immunodeficiency, growth retardation, and ultimately cancer predisposition. Microcephaly is a common outcome of genetic defects involving proteins of the centrosome and primary cilium (PC). This non-motile solitary organelle protrudes from the surface of all mammalian cells. Importantly, the centrosome converts into the basal body (BB) to enucleate the PC during the G0/G1 and regulates cytoskeleton dynamics to control PC length. Therefore, PC and DDR-related pathways converge in regulating neuronal progenitor cells (NPCs). Recently, we demonstrated that NBS1 depletion induces striking elongation and dysmorphisms of the PC in multiple cell models, coupled with defects in the Shh pathway, a PC-dependent mitogenic neurodevelopmental pathway involved in the proliferation and survival of NPCs. Given that, we speculated the existence of functional relationships between NBS1 and the Primary Cilium that might be responsible for the neurological phenotype observed in NBS.
In this study we evaluated: i) whether NBS1 depletion alters PC protein trafficking and Shh-pathway regulators; ii) the molecular mechanism through which NBS1 regulates the PC and iii) whether the most common hypomorphic mutation of the NBN gene, found in NBS patients, affects the PC. Our results demonstrated that NBS1-depleted cells have alterations of PC protein trafficking correlated with alterations of GliA/GliR ratio. We showed that the PC phenotype induced by NBS1 depletion is associated with alterations of the actin/tubulin dynamics, remarkably similar to that caused by cytochalasin D (CD), a strong inhibitor of actin polymerization. Indeed, treatments with CD did not further lengthen the PC in NBS1-depleted cells, suggesting that NBS1KO and CD may act through the same molecular mechanism/s. In addition, we demonstrated that the PC phenotype due to NBS1 depletion was counteracted by the exhaustion of soluble tubulin induced by Taxol. Coherently, CD impacted PC protein trafficking and the Shh pathway similarly to what was observed following the depletion of NBS1. Finally, we observed a significant increase in the PC length in human fibroblasts derived from an NBS patient (GM7166) compared to healthy controls, coupled with alterations both in PC protein trafficking and cytoskeleton dynamics.
Overall, our data provide crucial insights into the role of NBS1 on the molecular mechanisms involved in PC regulation, opening a possible reclassification of the NBS among the ciliopathies. Additionally, these results may contribute to explaining why genetic defects in centrosome/PC and DDR proteins may lead to the same neuronal phenotype.