Galactosylceramidase (GALC) enzymatic activity and psychosine accumulation in central and peripheral nervous system cells and tissues from wild-type and Twitcher mice

Globoid cell leukodystrophy (GLD, or Krabbe disease) is an autosomal recessive, neurodegenerative rare disease caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC). GALC degrades galactosylceramide, a major component of myelin, and other terminal b-galactose-containing sphingolipids, including the cytotoxic D-galactosylsphingosine (psychosine, PSY). GALC loss of function causes increased PSY levels in the neural tissues, leading to widespread degeneration of oligodendrocytes and Schwann cells and subsequent demyelination. Still little is known about the molecular mechanisms by which PSY imparts toxicity and no cure is currently available for GLD [Graziano et al., Gene 2015; Voccoli et al., Cell Death Dis 2014]. Unfortunately, the systemic GALC administration is not effective because of the presence of the blood brain barrier (BBB). The development of an enzyme replacement therapy (ERT) that takes advantage from the recent nanomedicine advancements to cross BBB would be one of the most promising options for curing GLD. In order to help in developing ERTs in the mouse GLD model, the Twitcher mouse (TWI), a study of GALC activity, PSY levels and viability in fibroblasts and different central and peripheral nervous system (CNS and PNS) cell types and tissues is here presented. We have set up cultures of primary wild-type (WT) and TWI fibroblasts and cells from CNS (i.e. astrocytes, oligodendrocytes and neurons) and characterized their GALC expression and activity by western-blotting and fluorimetric methods. These measurements were carried out both in cells lysates and in culture supernatants, allowing us to assess the level of enzyme exocytosis in different cell types. An analogous analysis is also reported for CNS and PNS tissues, such as: brain, optic and sciatic nerves. PSY quantification in WT and TWI homogenized brains, optic and sciatic nerves, and in different CNS cell types was performed by an LC-ESI-tandem-MS method. PSY accumulation in tissues was evaluated for TWI mice of different age. Moreover, data on cell viability of WT and TWI cells treated with different PSY concentrations are presented. Possible effect of autophagy modulators on cell viability will be finally discussed. A disturbance of autophagic pathways was indeed reported for many lysosomal storage disorders [Settembre et al., Autophagy 2008], and it has been linked to a deficiency of trafficking/processing of recombinant therapeutic enzymes, suggesting that autophagy dysfunction may have an impact in determining the efficacy of ERT.