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Biochim Biophys Acta. 2014 Apr;1842(4):665-75. doi: 10.1016/j.bbadis.2014.01.008. Epub 2014 Jan 24.

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

Author information

1
Unit of Biotechnology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
2
Unit of Oncology and Experimental Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
3
Unit of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
4
Department of Biology, University of Milan, Milan, Italy.
5
Research Unit on Bioactive Molecules (RUBAM), Department of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC), Spanish Council for Scientific Research (CSIC), Barcelona, Spain.
6
Department of Basic Biomedical Sciences, Unit of Human Anatomy and Histology, University of Bari, Bari, Italy; National Cancer Institute, Giovanni Paolo II, Bari, Italy.
7
Unit of Oncology and Experimental Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy. Electronic address: presta@med.unibs.it.

Abstract

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.

KEYWORDS:

Embryonic development; Galactosylceramidase; Krabbe disease; Sphingolipid; Zebrafish

PMID:
24463171
DOI:
10.1016/j.bbadis.2014.01.008
[Indexed for MEDLINE]
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