Molecular cloning and knockdown of galactocerebrosidase in zebrafish: new insights into the pathogenesis of Krabbe's disease.

The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galactosylceramide and from other sphingolipids. GALC deficiency is responsible for globoid cell leukodystrophy (GLD), or Krabbe's disease, an early lethal inherited neurodegenerative disorder characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system (CNS). The poor outcome of current clinical treatments calls for novel model systems to investigate the biological impact of GALC down-regulation and for the search of novel therapeutic strategies in GLD. Zebrafish (Danio rerio) represents an attractive vertebrate model for human diseases. Here, lysosomal GALC activity was demonstrated in the brain of zebrafish adults and embryos. Accordingly, we identified two GALC co-orthologs (named galca and galcb) dynamically co-expressed in CNS during zebrafish development. Both genes encode for lysosomal enzymes endowed with GALC activity. Single down-regulation of galca or galcb by specific antisense morpholino oligonucleotides results in a partial decrease of GALC activity in zebrafish embryos that was abrogated in double galca/galcb morphants. However, no psychosine accumulation was observed in galca/galcb double morphants. Nevertheless, double galca/galcb knockdown caused reduction and partial disorganization of the expression of the early neuronal marker neuroD and an increase of apoptotic events during CNS development. These observations provide new insights into the pathogenesis of GLD, indicating that GALC loss-of-function may have pathological consequences in developing CNS independent of psychosine accumulation. Also, they underscore the potentiality of the zebrafish system in studying the pathogenesis of lysosomal neurodegenerative diseases, including GLD.

Differentiation of rare leukodystrophies by post-mortem morphological and biochemical studies: female adrenoleukodystrophy-like disease and late-onset Krabbe disease.

Two 6-year-old patients with clinical signs of leukodystrophy had no nosological diagnoses in vivo. Neuropathological studies revealed scavenger cells to be clustered in perivascular regions of the demyelinated brains. Histochemical and ultrastructural details of the non-metachromatic storage macrophages suggested lipid storage and prompted a biochemical analysis of cerebral tissue. The detection of increased amounts of very long chain fatty acids in the cholesterol ester fraction from formalin tissue in one patient was consistent with a diagnosis of an adrenoleukodystrophy-like condition, while the marked reduction in beta-galactocerebrosidase activity in a frozen brain sample of the second patient indicate Krabbe disease. The diagnostic potential of post-mortem studies in rare leukodystrophies is addressed.