Abstract

A deficiency of arylsulfatase A (ASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disorder characterized by accumulation of sulfatide, a severe neurological phenotype and early death. The efficacy of enzyme replacement therapy (ERT) has previously been determined in ASA knockout (ASA-/-) mice representing the only available animal model for MLD. Repeated intravenous injection of human ASA (hASA) improved the nervous system pathology and function, but also elicited a progressive humoral immune response leading to treatment resistance, anaphylactic reactions, and high mortality. In contrast to ASA-/- mice, most MLD patients express mutant hASA which may entail immunological tolerance to substituted wildtype hASA and thus protect from immunological complications. To test this notion, a cysteine-to-serine substitution was introduced into the active site of the hASA and the resulting inactive hASA-C69S variant was constitutively expressed in ASA-/- mice. Mice with sub-to supranormal levels of mutant hASA expression were analyzed. All mice, including those showing transgene expression below the limit of detection, were immunologically unresponsive to injected hASA. More than 100-fold overexpression did not induce an overt new phenotype except occasional intralysosomal deposition of minor amounts of glycogen in hepatocytes. Furthermore, long-term, low-dose ERT reduced sulfatide storage in peripheral tissues and the central nervous system indicating that high levels of extracellular mutant hASA do not prevent cellular uptake and lysosomal targeting of substituted wildtype hASA. Due to the tolerance to hASA and maintenance of the MLD-like phenotype, the novel transgenic strain may be particularly advantageous to assess the benefit and risk of long-term ERT.

Highlights

  • Metachromatic leukodystrophy (MLD)is a lysosomal storage disease (LSD) which is caused by a functional deficiency of the lysosomal enzyme arylsulfatase A (ASA; E.C. 3.1.6.8.) [1]

  • The phenotype of ASA-/- mice recapitulates the early phases of metachromatic leukodystrophy (MLD), but does not proceed to the advanced stages, which are characterized by widespread demyelination, severe ataxia, blindness, quadriplegia, and loss of volitional movements [1,2]

  • In vitro Mutagenesis of the human ASA (hASA) cDNA The active site of eukaryotic sulfatases harbors a conserved cysteine residue which is essential for catalytic activity [20]

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Summary

Introduction

Is a lysosomal storage disease (LSD) which is caused by a functional deficiency of the lysosomal enzyme arylsulfatase A (ASA; E.C. 3.1.6.8.) [1]. ASA catalyzes the desulfation of the acidic sphingolipid 3-O-sulfogalactosylceramide (sulfatide) which is transported to the lysosomal compartment for degradation. Lack of ASA results in a block of sulfatide hydrolysis and the deposition of sulfatide in intralysosomal storage granules. Sulfatide storage in oligodendrocytes and Schwann cells leads to demyelination of the peripheral and central nervous system, a variety of rapidly deteriorating neurological symptoms and premature death. The phenotype of ASA-/- mice recapitulates the early phases of MLD, but does not proceed to the advanced stages, which are characterized by widespread demyelination, severe ataxia, blindness, quadriplegia, and loss of volitional movements [1,2]. Despite the relatively mild phenotype, the knockout mouse turned out to be a valuable model to test experimental therapies [3]

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