Dysregulated DNA methylation in the pathogenesis of Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A and subsequent accumulation of glycosphingolipids with terminal α-D-galactosyl residues. The molecular process through which this abnormal metabolism of glycosphingolipids causes multisystem dysfunction in Fabry disease is not fully understood. We sought to determine whether dysregulated DNA methylation plays a role in the development of this disease. In the present study, using isogenic cellular models derived from Fabry patient endothelial cells, we tested whether manipulation of α-galactosidase A activity and glycosphingolipid metabolism affects DNA methylation. Bisulfite pyrosequencing revealed that changes in α-galactosidase A activity were associated with significantly altered DNA methylation in the androgen receptor promoter, and this effect was highly CpG loci-specific. Methylation array studies showed that α-galactosidase A activity and glycosphingolipid levels were associated with differential methylation of numerous CpG sites throughout the genome. We identified 15 signaling pathways that may be susceptible to methylation alterations in Fabry disease. By incorporating RNA sequencing data, we identified 21 genes that have both differential mRNA expression and methylation. Upregulated expression of collagen type IV alpha 1 and alpha 2 genes correlated with decreased methylation of these two genes. Methionine levels were elevated in Fabry patient cells and Fabry mouse tissues, suggesting that a perturbed methionine cycle contributes to the observed dysregulated methylation patterns. In conclusion, this study provides evidence that α-galactosidase A deficiency and glycosphingolipid storage may affect DNA methylation homeostasis and highlights the importance of epigenetics in the pathogenesis of Fabry disease and, possibly, of other lysosomal storage disorders.

Assessing the role of glycosphingolipids in the phenotype severity of Fabry disease mouse model.

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 (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb3 isoforms (various fatty acids) and lyso-Gb3 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 Gb3 and lyso-Gb3 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 Gb3 and particular lyso-Gb3 analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb3 and lyso-Gb3 isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb3 massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb3 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 Gb3 or lyso-Gb3 may play major roles in the pathogenesis of Fabry disease, and that Gb3 and lyso-Gb3 are not responsible for the pathology in all tissues or cell types.

Priapism in a Fabry disease mouse model is associated with upregulated penile nNOS and eNOS expression.

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.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

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.

Molecular basis for globotriaosylceramide regulation and enzyme uptake in immortalized aortic endothelial cells from Fabry mice.

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.

Mannose receptor-mediated delivery of moss-made α-galactosidase A efficiently corrects enzyme deficiency in Fabry mice.

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.

Blocking hyperactive androgen receptor signaling ameliorates cardiac and renal hypertrophy in Fabry mice.

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.