Human iPSC-based neurodevelopmental models of globoid cell leukodystrophy uncover patient- and cell type-specific disease phenotypes

Abstract

21Globoid Cell Leukodystrophy (GLD, or Krabbe disease) is a rare lysosomal storage disease caused 22 by inherited deficiency of β-galactocerebrosidase (GALC). The build-up of psychosine and other 23 undegraded galactosylsphingolipids in the nervous system causes serious demyelination and 24 neurodegeneration. The molecular mechanisms of GLD are poorly elucidated in neural cells and whether 25 murine systems faithfully recapitulate critical aspects of the human disease is still to be defined. 26Here, we established a collection of GLD patient-specific induced pluripotent stem cell (iPSC) 27 lines. We differentiated iPSCs from two patients -bearing different disease-causing mutations -into 28 neural progenitors cells (NPCs) and their neuronal/glial progeny, assessing the impact of GALC deficiency 29 and lentiviral vector-mediated GALC rescue/overexpression by means of phenotypic, biochemical, 30 molecular, and lipidomic analysis. We show a progressive increase of psychosine during the 31 differentiation of GLD NPCs to neurons and glia. We report an early and persistent impairment of glial 32 and neuronal differentiation in GLD cultures, with peculiar differences observed in the two GLD lines. 33GLD cells display a global unbalance of lipid composition during the iPSC to neural differentiation and 34 early activation of cellular senescence, depending on the disease-causing mutation. Restoration of GALC 35 activity normalizes the primary pathological hallmarks and partially rescues the differentiation program 36 of GLD NPCs. 37Our results show that multiple mechanisms besides psychosine toxicity concur to CNS pathology 38 in GLD and highlight the need of a timely regulated GALC expression for proper lineage commitment and 39 differentiation of human NPCs. These findings have important implications for establishing tailored 40 cell/gene therapy strategies to enhance disease correction in GLD. 41 42