GBA1 under the microscope: exploring GBA1 mutations role in organelles’ dysfunction, and potential recovery through pharmacological chaperones

This thesis investigates the GBA1 gene, which encodes Glucocerebrosidase (GCase), a lysosomal enzyme critical for glucosylceramide and cholesterol metabolism. Over 300 pathogenic mutations have been identified, including L444P and N370S, which, in homozygous or compound heterozygous forms, cause Gaucher disease. In heterozygous forms, these mutations increase the risk of Parkinson’s disease. Mutant GCase misfolds, accumulating in the ER and triggering ER stress and the Unfolded Protein Response. Further downstream effects include lysosomal dysfunction, autophagic failure, Golgi fragmentation, and mitochondrial oxidative stress. The thesis aims to elucidate cellular defects caused by GBA1 mutations and explore therapeutic interventions. HEK293 11F cells (GBA1 knockdown) were analyzed using Western blotting to assess organelle health. Confocal and electron microscopy examined organelle abnormalities in fibroblasts from affected patients. Site-directed mutagenesis was employed to create plasmids harboring N370S and L444P mutations for advanced model development. Additionally, a plasmid combining GBA1 with pH-sensitive mKeima was generated to track GCase trafficking. Finally, potential pharmacological chaperones, compounds 11 and 12 from the University of Firenze, were tested on patient fibroblasts to restore GCase activity.