CEP120-mediated KIAA0753 recruitment onto centrioles is required for timely neuronal differentiation and germinal zone exit in the developing cerebellum

Joubert syndrome (JS) is a recessive ciliopathy in which all affected individuals have congenital cerebellar vermis hypoplasia. Here, we report that CEP120, a JS-associated protein involved in centriole biogenesis and cilia assembly, regulates timely neuronal differentiation and the departure of granule neuron progenitors (GNPs) from their germinal zone during cerebellar development. Our results show that depletion of Cep120 perturbs GNP cell cycle progression, resulting in a delay of cell cycle exit in vivo. To dissect the potential mechanism, we investigated the association between CEP120 interactome and the JS database and identified KIAA0753 (a JS-associated protein) as a CEP120-interacting protein. Surprisingly, we found that CEP120 recruits KIAA0753 to centrioles, and that loss of this interaction induces accumulation of GNPs in the germinal zone and impairs neuronal differentiation. Importantly, the replenishment of wild-type CEP120 rescues the above defects, whereas expression of JS-associated CEP120 mutants, which hinder KIAA0753 recruitment, does not. Together, our data reveal a close interplay between CEP120 and KIAA0753 for the germinal zone exit and timely neuronal differentiation of GNPs during cerebellar development, and mutations in CEP120 and KIAA0753 may participate in the heterotopia and cerebellar hypoplasia observed in JS patients.

CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome.

Cerebellum and Prematurity: A Complex Interplay Between Disruptive and Dysmaturational Events

The cerebellum plays a critical regulatory role in motor coordination, cognition, behavior, language, memory, and learning, hence overseeing a multiplicity of functions. Cerebellar development begins during early embryonic development, lasting until the first postnatal years. Particularly, the greatest increase of its volume occurs during the third trimester of pregnancy, which represents a critical period for cerebellar maturation. Preterm birth and all the related prenatal and perinatal contingencies may determine both dysmaturative and lesional events, potentially involving the developing cerebellum, and contributing to the constellation of the neuropsychiatric outcomes with several implications in setting-up clinical follow-up and early intervention.

EMBR-10. INOSITOL TREATMENT INHIBITS MEDULLOBLASTOMA THROUGH SUPPRESSION OF EPIGENETIC-DRIVEN METABOLIC ADAPTATION

Medulloblastoma (MB) is the most common paediatric malignant brain tumour and is classified into four distinct molecular subgroups (WNT, SHH, G3 and G4), each of them further subdivided into subtypes with different prognosis and responses to therapy. Deregulation of chromatin modifier genes plays an essential role in MB, particularly in the G4 subgroup, the least understood of all subgroups, despite being the most common and associated with poor prognosis. A BMI1High; CHD7Low molecular signature identifies patients with poor survival within this subgroup. We show that BMI1High; CHD7Low mediates a novel epigenetic regulation of inositol metabolism in both G4 MB cells and patients. These tumours display hyperactivation of the AKT/mTOR pathway which leads to energetic rewiring characterized by enhanced glycolytic capacity and reduced mitochondrial function. We demonstrate that inositol administration counteracts this metabolic alteration, impairs MB proliferation in vitro and significantly extends survival in an in vivo pre-clinical model. Moreover, inositol synergises with cisplatin, a chemotherapy agent currently used in MB treatment, enhancing its therapeutic effect in vivo. Importantly, cerebellar neural stem cells bearing the BMI1High; CHD7Low signature do not show metabolic adaptation and are thus resistant to inositol treatment, highlighting a fundamental difference between normal and neoplastic metabolism in the developing cerebellum. In summary, we have identified an actionable vulnerability in a pre-clinical setting modelling a molecularly defined group of MB patients, the translational value of which can now be explored in signature-matched clinical trials in MB.

Ten-eleven translocation protein 1 modulates medulloblastoma progression

Background Medulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes. Results We investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened (SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes. Conclusions These results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

IMMUNOHISTOCHEMICAL STUDY OF CALBINDIN IN THE DEVELOPING CEREBELLUM OF THE RAT

Background. Calbindin is a calcium-binding protein that supports calcium homeostasis for the normal functioning of neurons. Purpose. To study the distribution of immunoreactivity of calbindin-D28K in the structures of the developing cerebellum of the rat.Material and methods. The study was performed on 16 outbred white rats of different age groups: 2-, 7-, 15-days (early postnatal period), 45-days (puberty period). The cerebellum samples were taken and fixed in zinc-ethanol-formaldehyde for immunohistochemistry. Calbindin-D28K immunoreactivity was determined on paraffin sections using primary polyclonal rabbit antibodies.Results. In the cerebellar cortex, calbindin immunoreactivity was detected on the 2nd day after development of Purkinje cells (PC) in their perikaryons, and by the 15th day in their dendrites and it did not change by the 45th day. In all terms of the study in PC, it was detected not only in the cytoplasm, but also in their nucleus. In the granular layer, calbindin immunoreactivity decreased in rats in postnatal ontogenesis, however, in adult rats, some neurons were moderately immunopositive. Among them, from the 15th day after birth, the calbindin-immunoreactive afferent nerve fibers running in the white matter were detected. There were no significant differences in the distribution of calbindin between the lobes of the cerebellum of different phylogenetic age. Conclusions. Considering that the expression of сalbindin-D28k is detected throughout the entire period of development of Purkinje cells, as well as its physiological role in maintaining the function and homeostasis of calcium in them, it can be concluded that сalbindin-D28k is a valuable marker for the morphofunctional characteristics of PC in the developing and adult cerebellum of rats in normal and pathological conditions.