Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: In vivo characterization of KCASH2 in the Central Nervous System: the KCASH2 knock out model.

KCASH2 was discovered in 2011 as a member of the KCASH family which acts as negative regulators of the Hedgehog signaling pathway during cerebellar development and differentiation (De Smaele, Di Marcotullio et al. 2011). KCASH2, thanks to its BTB/POZ domain, acts mediating the binding between Cul3 ubiquitin ligase and HDAC1 deacetylase, and the subsequent HDAC1 proteasomal degradation. The consequent loss of HDAC1 therefore maintains Gli1, the main transcription factor of the Hh pathway, in an acetylated state, decreasing its transcriptional activity. Since its discovery KCASH2 has been observed to act as a tumour suppressor in Hedgehog-dependent Medulloblastoma. coherently KCASH2 loss leads to persistent Hh activity and enhanced tumour growth. (De Smaele, Di Marcotullio et al. 2011) (Spiombi, Angrisani et al. 2019). Moreover, KCASH2 was observed downmodulated in many MB samples. Hedgehog signaling during embryogenesis and in adult life must be finely regulated and its deregulation may lead to cerebellar tumorigenesis but also to other pathologies, from basal cell carcinoma to abnormalities in limb development. To study the in vivo implications of KCASH2 in Hh signalling, development and tumorigenesis we generated the KCASH2 KO mouse model, and initiated its characterization. During this work, we confirmed the role of KCASH2 as a negative regulator of the Hh pathway in cerebellar development. Indeed, we found that in the absence of KCASH2 during the early stages of GCPs development there is an accumulation of HDAC1 and consequently an increase of Gli1 levels. GCPs respond to Hh up-regulation by increasing their proliferation rate resulting in a Hedgehog-like altered cerebellar phenotype in adult mice. In addition, we also identified unexpected new sites of expression and new functions for KCASH2. We demonstrate here KCASH2 expression in other cells of the cerebellum, the Purkinje cells (PC), and also in other cells of the brain and spinal cord. In particular, KCASH2 loss determines in PC switch from low HDAC1 low to High HDAC1 levels (an unexpected finding). KCASH2 is also expressed in brain and Spinal cord astrocytes, and its loss seems to determine a decrease in SHh secretion favouring their switch from quiescent to reactive astrocytes. Reduced production of astrocyte-derived N-SHh causes in the Endothelial cells (EC) (which constitute the blood vessels) an increase of levels of the Tight Junction key protein Claudin 5, decreasing Blood Brain Barrier permeability. This new finding warrants further work on the KCASH2KO model, with interesting potential applications on the study of neuronal tissue development, regeneration, tumorigenesis and on blood brain barrier functions.