Insights into MLC pathophysiology: a biochemical and structural approach

[eng] Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by macrocephaly and white matter vacuolation. The pathogenesis of the disease is suggested to be caused by an impaired water and ionic homeostasis by glial cells. MLC is caused by mutations in MLC1, GLIALCAM, GPRC5B and AQP4. MLC1 and GLIALCAM encode for membrane proteins that form a complex in astrocytes, whose exact function remains unclear. Following studies identified the G- protein-coupled receptor GPRC5B as an important member of the GlialCAM/MLC1 interactome and relevant to the regulation of related physiological processes. One of the main objectives of this thesis is to determine the role of GPRC5B and its signalling activity related to the pathophysiology of MLC. We determine that MLC1 has a negative modulatory effect on GPRC5B signalling pathways. Besides, we propose that mutations in GLIALCAM that encode for residues located in GlialCAM IgC2 domain are pathogenic due to the increased stability of GlialCAM oligomeric structures. Moreover, we give evidence that GlialCAM endocytosis is mediated by GPRC5B. We also study the recent identified mutations in GPRC5B known to cause MLC and observe a resistance to internalization resulting in the same outcome as those GlialCAM IgC2 mutations. Another objective of this thesis is to obtain the tridimensional structure of MLC1. We have achieved a preliminary structure of homo-trimeric MLC1 at a resolution of 7 Å, approximately. At the same time, we have developed nanobodies that specifically recognize MLC1 to help orientate particles during cryo-electron microscopy (cryo-EM).