Titolo della tesi: Dissecting the essential function of NBS1 on Sonic Hedgehog-dependent cerebellar development and transformation
Neurodevelopmental disorders (e.g. microcephaly and ataxia) are often associated with DNA Damage Response (DDR)-defective syndromes. However, why the nervous system is extremely vulnerable to defects in DDR-related genes is currently unknown.
We recently demonstrated that NBS1, a member of the MRE11/RAD50/NBS1 (MRN) complex, one of the first actors involved in DDR, regulates the morphology and functionality of the primary cilium (PC). Specifically, its depletion leads to a striking elongation and dysmorphism of the PC associated with a strong impairment of the Sonic Hedgehog (SHH) pathway both in vivo and ex vivo. PC is a signaling organelle essential for the regulation of the SHH pathway during cerebellar histogenesis, organized by the centrosome converted in the basal body (BB) during ciliogenesis.
Notably, many DDR proteins, including NBS1, localize at the centrosome, and alterations in centrosome functionality are associated with neurodevelopmental diseases, suggesting a functional link between DDR-related factors and centrosomes. Since we found that DNA damage accumulation, induced by genotoxic drugs, does not affect PC length or morphology, we hypothesized that NBS1 could play its role in PC regulation in an uncanonical manner, directly at the centrosome/BB by the interaction with BB/PC-related proteins.
In this study, we: i) characterized the effect of the NBS1 knock-out (KO) on the SHH-dependent development and transformation of the cerebellum and ii) proposed a strategy to study the new role of NBS1 on primary ciliogenesis by exploring its interactors at the centrosomes.
To achieve our first objective, we generated and characterized a new mouse model where the KO of Nbn (NBS1 in mouse) is restricted to the population of the cerebellar granule cell progenitors (GCPs) both in a wild-type (Nbn-GCPΔ) and medulloblastoma (MB) (Ptch1/Nbn-GCPΔ) cancer-prone background. Our findings indicated that Ptch1/Nbn-GCPΔ mice had reduced thickness of external granular layer (EGL)/internal granular layer (IGL) in the anterior lobes while developed tumors in the posterior ones due to an incomplete cleavage of the Nbn allele in that cerebellar region. Similarly, Nbn-GCPΔ mice displayed overall cerebellar hypoplasia, with a more pronounced reduction in EGL/IGL thickness in the anterior lobes. Additionally, we found that the architectural defects observed in the Nbn-GCPΔ mice were associated with a significant impairment in the proliferation of the GCPs.
Therefore, this initial phase of our study demonstrated that the depletion of NBS1 specifically within the GCPs population is sufficient to disrupt SHH-driven proliferation and transformation in vivo.
Relative to the second aim, we first confirmed the presence of NBS1 not only at the centrosome but also at the BB of the PC by multiple approaches and in several cell lines. Second, we developed an approach to detect the interactions of NBS1 at the centrosome, hardly accessible to the standard methods, using the innovative proximity-dependent biotinylation (BioID) technique coupled with biochemical centrosome enrichment by sucrose gradient fractionation.
In conclusion, on one side our study demonstrates an essential role of NBS1 in the SHH-dependent development and tumorigenesis of the cerebellum, strongly supporting the possibility that NBS1 alteration might lead to neurological defects impacting on PC-dependent signaling pathways. On the other side, it provided a new technical approach to go deeper into the uncanonical role of NBS1 at the centrosome/PC.
We believe that our innovative study will improve our understanding on the molecular mechanisms that define the regulatory role of NBS1 on the PC/SHH pathway and could help to understand the functional link between centrosomes, DDR proteins and neuronal disease.