Search

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb(3)), globotriaosylsphingosine (lyso-Gb(3)), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb(3) isoforms (various fatty acids) and lyso-Gb(3) analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb(3) and lyso-Gb(3) levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb(3) and particular lyso-Gb(3) analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb(3) and lyso-Gb(3) isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb(3) massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb(3) accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb(3) or lyso-Gb(3) may play major roles in the pathogenesis of Fabry disease, and that Gb(3) and lyso-Gb(3) are not responsible for the pathology in all tissues or cell types.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered. Electronic supplementary material The online version of this article (doi:10.1007/s10545-015-9886-9) contains supplementary material, which is available to authorized users.

Knockout mouse model of Fabry disease (Ohshima, 1997) is widely used, and has been proven an invaluable tool to investigate disease pathogenesis and therapies. However, the importance of genetic background of this mouse line has been underestimated, and in many publications WT control mice were chosen incorrectly. Our aim was to examine the effects of genetic background on biochemical and functional abnormalities of Fabry mice. We characterized Fabry mice in B6/129 mixed background (~75% B6) and Fabry mice on a purely C57BL/6 strain (10 generations backcrossing), compared with WT mice on an identical genetic background. Both Fabry-B6/129 and Fabry-B6 developed progressive cardiac and renal hypertrophy and impaired thermosensation. However, Fabry-B6/129 exhibited earlier onset and more severe phenotypes than Fabry-B6. Cardiac and/or renal hypertrophy appeared at age of 6 months in Fabry-B6/129, but at 12 months in Fabry-B6. At 12 months, kidney-to-body weight ratio increased by 50% in Fabry-B6/129 (vs. WT), but only 15% in Fabry-B6. The onset of the thermosensation abnormality was at age 2 months in Fabry-B6/129 and 7 months in Fabry-B6. Cardiac expression of hypertrophy marker, atrial natriuretic peptide (ANP), was upregulated in Fabry-B6/129, but not in Fabry-B6. Despite these differences, globotriaosylceramide (Gb3) and lyso-Gb3 levels in the heart, kidney and dorsal root ganglia were similar between Fabry-B6/129 and Fabry-B6. However, Gb3 level in liver (organ unaffected in Fabry disease) was twofold higher in Fabry-B6/129 compared to Fabry-B6. Additionally, several parameters including body weight, organ weight, kidney Gb3 and cardiac ANP level were different between WT-B6/129 and WT-B6. In conclusion: 1) genetic background has a significant impact on the severity and onset of phenotypes in Fabry mice 2) the lack of correlation between phenotype and Gb3/lyso-Gb3 levels in disease-relevant organs suggests that the effect of genetic background on the phenotype is determined by other genetic and epigenetic factors.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3). Vascular endothelial cells may play important roles in disease pathogenesis, and are one of the main target cell types in therapeutic interventions. In this study, we generated immortalized aortic endothelial cell lines from a mouse model of Fabry disease. These cells retained endothelial cell-specific markers and functions. Gb3 expression level in one of these clones (referred to as FMEC2) was highly susceptible to culture media, and appeared to be regulated by glucosylceramide synthase. Results also showed that Gb3 could be upregulated by hydrocortisone. FMEC2 express the mannose 6-phosphate receptor and sortilin but not the mannose receptor. Uptake studies suggested that sortilin plays a role in the binding and internalization of mammalian cell-produced α-galactosidase A. Moss-aGal (a plant-made enzyme) was endocytosed by FMEC2 via a receptor other than the aforementioned receptors. In conclusion, this study suggests that glucosylceramide synthase and hydrocortisone may play important roles in modulating Gb3 levels in Fabry mouse aortic endothelial cells, and that endocytosis of recombinant α-galactosidase A involves a combination of multiple receptors depending on the properties of the enzyme.

Fabry disease is caused by deficient activity of lysosomal enzyme α-galactosidase A. The enzyme deficiency results in intracellular accumulation of glycosphingolipids, leading to a variety of clinical manifestations including hypertrophic cardiomyopathy and renal insufficiency. The mechanism through which glycosphingolipid accumulation causes these manifestations remains unclear. Current treatment, especially when initiated at later stage of the disease, does not produce completely satisfactory results. Elucidation of the pathogenesis of Fabry disease is therefore crucial to developing new treatments. We found increased activity of androgen receptor (AR) signaling in Fabry disease. We subsequently also found that blockade of AR signaling either through castration or AR-antagonist prevented and reversed cardiac and kidney hypertrophic phenotype in a mouse model of Fabry disease. Our findings implicate abnormal AR pathway in the pathogenesis of Fabry disease and suggest blocking AR signaling as a novel therapeutic approach.