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Study of the molecular bases underlying cerebellar defects with emphasis on protein N-glycosylation impairment

Abstract : Cerebellar defects encompass a group of rare diseases affecting cerebellar functioning. The prevalence of these defects is estimated to be 26 in 10000 children in Europe. These diseases lead to movement disorders (ataxia) and are frequently associated with intellectual disability, life-threatening conditions that help affected patients from coping with a normal daily life. For most of these conditions, only supportive treatment is available. Besides genetic diagnose, helpful when facing new pregnancies, an in-deep understanding of the physiopathology underlying the disorder is necessary for future therapeutics. My thesis project had as objective to improve the genetic diagnose of cerebellar defects and understanding the physiopathology behind one of the more prevalent cause of cerebellar defects: disruption of protein N-glycosylation. Disruption of protein N-glycosylation causes Congenital Disorders of Glycosylation (CDG), multisystemic disorders with severe neurological disorders. Early-onset cerebellar atrophy and hypoplasia are frequently observed, especially in CDG cases with SRD5A3 mutations. To understand how a general N-glycosylation defect affects cerebellar development, we generated a cerebellum-specific Srd5a3 conditional KO mouse. This model recapitulates the human defect with abnormal N-glycosylation, cerebellar hypoplasia and motor coordination impairment. Careful histological evaluation of the cerebellum proved some granule cells to be unable to initiate their final migration during cerebellar development. By combining a proteomic and a glycoproteomic approach, we showed that a defect in N-glycosylation has a variable impact depending on the number of N-glycosylation sites on each protein. The more N-glycosylation sites that a protein has, the more sensitive it is to hypoglycosylation and/or degradation in a CDG context. Our data suggest the heavily N-glycosylated cell adhesion molecules with immunoglobulin domains (IgSF-CAMs) to be highly sensitive to the glycosylation defect. Using in vitro live granule cells imaging, we identified an IgSF-CAM-dependant neurite extension defect. This defect is linked to impaired glycosylation and functioning of L1CAM and NrCAM. We next evaluated if the defect was conserved in human neurons. To investigate that possibility we generated SRD5A3-/- hiPSCs that were further differentiated towards cortical neurons. Human neuron-like cells recapitulate the biochemical defect in mouse (e.g. L1CAM and NrCAM hypoglycosylation). This finding expands our conclusions to the whole human brain. Finally, using electron microscopy we could identify disrupted cerebellar parallel fiber organization in the mouse mutant, consistent with the established role of numerous IgSF-CAMs in axon guidance. Our results provide important evidence into the molecular mechanism underlying cerebellar sensitivity to an N-glycosylation impairment. Moreover, we show how defects in N-glycosylation will primarily affect cell adhesion. Our work also provides new evidence for the critical importance of the multiple N-glycosylation of IgSF-CAMs for their stability and functionality during mammalian brain development.
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Daniel Medina-Cano. Study of the molecular bases underlying cerebellar defects with emphasis on protein N-glycosylation impairment. Medical Imaging. Université Sorbonne Paris Cité, 2018. English. ⟨NNT : 2018USPCB054⟩. ⟨tel-02493474⟩

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