We employed induced pluripotent stem cell (iPSC)-derived neurons obtained from Friedreich ataxia (FRDA) patients and healthy subjects, FRDA neurons and CT neurons, respectively, to unveil phenotypic alterations related to frataxin (FXN) deficiency and investigate if they can be reversed by treatments that upregulate FXN. FRDA and control iPSCs were equally capable of differentiating into a neuronal or astrocytic phenotype. FRDA neurons showed lower levels of iron–sulfur (Fe–S) and lipoic acid-containing proteins, higher labile iron pool (LIP), higher expression of mitochondrial superoxide dismutase (SOD2), increased reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels, and enhanced sensitivity to oxidants compared with CT neurons, indicating deficient Fe–S cluster biogenesis, altered iron metabolism, and oxidative stress. Treatment with the benzamide HDAC inhibitor 109 significantly upregulated FXN expression and increased Fe–S and lipoic acid-containing protein levels, downregulated SOD2 levels, normalized LIP and ROS levels, and almost fully protected FRDA neurons from oxidative stress-mediated cell death. Our findings suggest that correction of FXN deficiency may not only stop disease progression, but also lead to clinical improvement by rescuing still surviving, but dysfunctional neurons.

Globoid cell leukodystrophy (GLD) is a rare neurodegenerative lysosomal storage disease caused by an inherited deficiency of b-galactocerebrosidase (GALC). GLD pathogenesis and therapeutic correction have been poorly studied in patient neural cells. Here, we investigated the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in induced pluripotent stem cell (iPSC)derived neural progenitors and neuronal/glial progeny obtained from two GLD patients. GLD neural progeny displayed progressive psychosine storage, oligodendroglial and neuronal defects, unbalanced lipid composition, and early activation of cellular senescence, depending on the disease-causing mutation. The partial rescue of the neural differentiation program upon GALC reconstitution and psychosine clearance suggests multiple mechanisms contributing to neural pathology in GLD. Also, the pathological phenotype associated to supraphysiological GALC levels highlights the need of regulated GALC expression for proper human neural commitment/differentiation. These data have important implications for establishing safe therapeutic strategies to enhance disease correction of GLD.

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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

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