Genistein reduces lysosomal storage in peripheral tissues of mucopolysaccharide IIIB mice

Abstract

Mucopolysaccharidosis type IIIB (Sanfilippo syndrome) is a lysosomal storage disease caused by a genetic defect in the production of α-N-acetylglucosaminidase. This results in lysosomal and extracellular accumulation of the undegraded glycosaminoglycan (GAG) substrate, heparan sulphate. Affected patients show progressive CNS degeneration characterised by mental retardation, hyperactivity and seizures, with death usually in the mid teens to early twenties. Visceral organ storage is also present but is relatively mild compared to other MPS diseases storing similar substrates. No treatments currently exist for MPS IIIB.

Genistein is a broad spectrum protein tyrosine kinase inhibitor which acts on several different growth factor receptors, notably EGF and IGF receptors, both of which are important for proteoglycan synthesis. Recent work has shown that genistein can reduce GAG synthesis in patients’ fibroblasts in vitro and there is evidence in patients to suggest that it may be an effective substrate reduction therapy agent for MPS III. Here we have tested the dose responses of MPS IIIB mice to daily sub-chronic dosing of genistein in half log increments compared to carrier over 8 weeks.

We show clear reductions in liver lysosome compartment size in both sexes and significant dose dependent improvements in total liver GAGs and hair morphology in male MPS IIIB animals following genistein treatment. Male MPS IIIB mice exhibited considerably more liver storage than females and responded better to treatment. No changes in total GAGs, lysosomal size or reactive astrogliosis in the brain cortex were observed after 8 weeks of treatment despite evidence that genistein can cross the blood brain barrier. This is the first demonstration of genistein treatment in MPS models in vivo.

Introduction

Mucopolysaccharidosis III, or Sanfilippo syndrome, consists of 4 lysosomal storage diseases, all caused by a genetic defect in the production of specific lysosomal enzymes. Defects in these enzymes result in lysosomal and extracellular storage of the glycosaminoglycan (GAG) heparan sulphate (HS). Affected patients share very similar symptoms including progressive CNS degeneration characterised by mental retardation, hyperactivity and seizures, with death usually in the second or third decade. Visceral organ storage is also present but is relatively mild compared to other MPS diseases storing GAGs [1].

Enzyme replacement therapy and even haematopoietic stem cell transplantation (HSCT) are effective strategies for treating other mucopolysaccharide diseases [2], [3], [4]. In the case of enzyme replacement therapy, the ability of cells to take up exogenous enzyme via mannose 6 phosphate receptors is exploited. The main problem with this approach is delivery of enzyme to cells in the brain, which is hampered by the presence of the blood brain barrier, blocking free passage of most lysosomal enzymes or of corrected cells. This limits the utility of enzyme replacement therapy to milder diseases without CNS involvement. Haematopoietic cell therapy relies on intra-cerebral transport of microglial cells from the haematopoietic system, and although it is effective at ameliorating CNS degeneration for mucopolysaccharidosis type I, it has not proven to be effective in MPS III [5].

Treatments aimed at the cellular reduction of substrates accrued in lysosomal storage disorders either by blocking an earlier step in the catabolism of the substrate, or rerouting the affected metabolic pathway by altered signalling or gene silencing, are thus very attractive, particularly since other enzymes in the lysosomal pathway are often upregulated to compensate for the deficiency. Assuming that a drug can cross the blood brain barrier, there is potential to target every cell in the body and thus have more opportunity to correct the neuronal aspect of the disorders. This approach has been used successfully in glycosphingolipid storage disorders to reduce GM 1 and 2 gangliosides in Sandhoff disease, Tay-Sachs and Gaucher disease (reviewed in [6]). In mucopolysaccharide diseases, gentamycin has been shown to improve stop codon read through in cells from MPS I patients and thus boost enzyme levels [7], whilst rhodamine B, a non-specific inhibitor of GAG chain elongation has been shown to reduce GAG accumulation [8], and improve spatial learning and memory in MPS IIIA mice [9]. Other inhibitors of GAG synthesis have been described, such as 4-deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-d-glucose and 2-acetamido-2-deoxy-d-galactose [10] but their availability may be limited and there are problems with long-term toxicity of drugs such as gentamycin or rhodamine B.

Recent studies have shown that one of the components of soy extract, genistein (4′,5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibits synthesis of glycosaminoglycans (GAGs) in cultures of fibroblasts from MPS patients (types I, II, IIIA and IIIB) [11].

Prolonged cultivation of MPS fibroblasts in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells. Genistein has been shown to inhibit the tyrosine specific protein kinase activity of epidermal growth factor (EGF) receptor, which is required for full expression of genes coding for enzymes involved in GAG production [12]. This has been shown to be at least partly responsible for genistein’s effect on reducing GAG storage in patient fibroblasts [13]. Genistein has also been shown to cross the blood brain barrier at levels of approximately 10% of those attained in blood [14], making it an ideal candidate for substrate reduction therapy in MPS III.

We have used the well characterised murine MPS IIIB model created by a targeted disruption in the α-N-acetylglucosaminidase (Naglu) gene [15] to investigate the potential of genistein as a therapy for MPS IIIB. In this dose response study, we have tested half log incremental doses of genistein up to the maximum tolerated dose in both wild-type (WT) and MPS IIIB mice of both sexes against carrier alone. This has allowed us to evaluate the most efficacious doses of genistein for treating MPS IIIB mice, the shape of the dose response curve and any sex related differences between mice.