Usefulness of antibody-drug conjugate as preconditioning for hematopoietic stem cell-targeted gene therapy in wild-type and Fabry disease mouse models.

BACKGROUND: Fabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice. METHODS: After preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed. RESULTS: In the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs. CONCLUSIONS: Antibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD.

Mass cytometry reveals atypical immune profile notably impaired maturation of memory CD4 T with Gb3-related CD27 expression in CD4 T cells in Fabry disease.

Fabry disease (FD) is an X-linked disease characterized by an accumulation of glycosphingolipids, notably of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) leading to renal failure, cardiomyopathy, and cerebral strokes. Inflammatory processes are involved in the pathophysiology. We investigated the immunological phenotype of peripheral blood mononuclear cells in Fabry patients depending on the clinical phenotype, treatment, Gb3, and lysoGb3 levels and the presence of anti-drug antibodies (ADA). Leucocytes from 41 male patients and 20 controls were analyzed with mass cytometry using both unsupervised and supervised algorithms. FD patients had an increased expression of CD27 and CD28 in memory CD45- and CD45 + CCR7-CD4 T cells (respectively p < 0.014 and p < 0.02). Percentage of CD45RA-CCR7-CD27 + CD28+ cells in CD4 T cells was correlated with plasma lysoGb3 (r = 0.60; p = 0.0036) and phenotype (p < 0.003). The correlation between Gb3 and CD27 in CD4 T cells almost reached significance (r = 0.33; p = 0.058). There was no immune profile associated with the presence of ADA. Treatment with agalsidase beta was associated with an increased proportion of Natural Killer cells. These findings provide valuable insights for understanding FD, linking Gb3 accumulation to inflammation, and proposing new prognostic biomarkers.

Fabry disease in W162C mutation: a case report of two patients and a review of literature.

BACKGROUND: Fabry disease is a multisystemic disorder characterized by deposition of globotriaosylceramide (Gb3) and its deacylated form in multiple organs, sometimes localized in specific systems such as the nervous or cardiovascular system. As disease-modifying therapies are now available, early diagnosis is paramount to improving life quality and clinical outcomes. Despite the widespread use of non-invasive techniques for assessing organ damage, such as cardiac magnetic resonance imaging (MRI) for patients with cardiac disease, organ biopsy remains the gold standard to assess organ involvement. CASE PRESENTATION: The cases of two patients, father and daughter with a W162C mutation, are described. The father presented with late-onset, cardiac Fabry disease, subsequently developing systolic dysfunction and heart failure. His daughter, while asymptomatic and with normal cardiac assessment (except for slightly reduced native T1 values by cardiac MRI), had already initial myocyte Gb3 deposits on the endomyocardial biopsy, allowing her to start therapy precociously and potentially modifying the course of her disease. A review of the literature concerning the W162C mutation is then provided, showing that it is usually associated to classic, multisystemic Fabry disease rather than the cardiac-restricted form as in these two cases. CONCLUSIONS: Three main points can be concluded from this report. First, the W162C mutation can present with a more variegate phenotype than that predicted on a molecular basis. Second, endomyocardial biopsy was shown in this case to precede non-invasive investigation in determining organ involvement, justifying further studies on this potentially reliable technique, Third, difficulties can arise in the management of asymptomatic female carriers.

2024 Update of the TSOC Expert Consensus of Fabry Disease.

As an X-linked inherited lysosomal storage disease that is caused by α-galactosidase A gene variants resulting in progressive accumulation of pathogenic glycosphingolipid (Gb3) accumulation in multiple tissues and organs, Fabry disease (FD) can be classified into classic or late-onset phenotypes. In classic phenotype patients, α-galactosidase A activity is absent or severely reduced, resulting in a more progressive disease course with multi-systemic involvement. Conversely, late-onset phenotype, often with missense variants (e.g., IVS4+919G>A) in Taiwan, may present with a more chronic clinical course with predominant cardiac involvement (cardiac subtype), as they tend to have residual enzyme activity, remaining asymptomatic or clinically silent during childhood and adolescence. In either form, cardiac hypertrophy remains the most common feature of cardiac involvement, potentially leading to myocardial fibrosis, arrhythmias, and heart failure. Diagnosis is established through α-galactosidase enzyme activity assessment or biomarker analyisis (globotriaosylsphingosine, Lyso-Gb3), advanced imaging modalities (echocardiography and cardiac magnetic resonance imaging), and genotyping to differentiate FD from other cardiomyopathy. Successful therapeutic response relies on early recognition and by disease awareness from typical features in classic phenotype and cardiac red flags in cardiac variants for timely therapeutic interventions. Recent advances in pharmacological approach including enzyme replacement therapy (agalsidase alfa or beta), oral chaperone therapy (migalastat), and substrate reduction therapy (venglustat) aim to prevent from irreversible organ damage. Genotype- and gender-based monitoring of treatment effects through biomarker (Lyso-Gb3), renal assessment, and cardiac responses using advanced imaging modalities are key steps to optimizing patient care in FD.

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells.

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins  that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

Gene expression profile and injury sites in mice treated with Shiga toxin 2 and lipopolysaccharide as a Shiga toxin-associated hemolytic uremic syndrome model.

Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) contribute to the development of hemolytic uremic syndrome (HUS). Mouse models of HUS induced by LPS/Stx2 have been used for elucidating HUS pathophysiology and for therapeutic development. However, the underlying molecular mechanisms and detailed injury sites in this model remain unknown. We analyzed mouse kidneys after LPS/Stx2 administration using microarrays. Decreased urinary osmolality and urinary potassium were observed after LPS/Stx2 administration, suggestive of distal nephron disorders. A total of 1,212 and 1,016 differentially expressed genes were identified in microarrays at 6 h and 72 h after LPS/Stx2 administration, respectively, compared with those in controls. Ingenuity pathway analysis revealed activation of TNFR1/2, iNOS, and IL-6 signaling at both time points, and inhibition of pathways associated with lipid metabolism at 72 h only. The strongly downregulated genes in the 72-h group were expressed in the distal nephrons. In particular, genes associated with distal convoluted tubule (DCT) 2/connecting tubule (CNT) and principal cells of the cortical collecting duct (CCD) were downregulated to a greater extent than those associated with DCT1 and intercalated cells. Stx receptor globotriaosylceramide 3 (Gb3) revealed no colocalization with DCT1-specific PVALB and intercalated cell-specific SLC26A4 but did present colocalization with SLC12A3 (present in both DCT1 and DCT2), and AQP2 in principal cells. Gb3 localization tended to coincide with the segment in which the downregulated genes were present. Thus, the LPS/Stx2-induced kidney injury model represents damage to DCT2/CNT and principal cells in the CCD, based on molecular, biological, and physiological findings.

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

A novel unbiased method reveals progressive podocyte globotriaosylceramide accumulation and loss with age in females with Fabry disease.

While females can suffer serious complications of Fabry disease, most studies are limited to males to avoid confounding by mosaicism. Here, we developed a novel unbiased method for quantifying globotriaosylceramide (GL3) inclusion volume in affected podocytes (F+) in females with Fabry disease independent of mosaicism leading to important new observations. All podocytes in male patients with Fabry are F+. The probability of observing random profiles from F+ podocytes without GL3 inclusions (estimation error) was modeled from electron microscopic studies of 99 glomeruli from 40 treatment-naïve males and this model was applied to 28 treatment-naïve females. Also, podocyte structural parameters were compared in 16 age-matched treatment-naïve males and females with classic Fabry disease and 11 normal individuals. A 4th degree polynomial equation best described the relationship between podocyte GL3 volume density and the estimation error (R2 =0.94) and was confirmed by k-fold cross-validation. In females, this model showed that age related directly to F+ podocyte GL3 volume (correlation coefficient (r = 0.54) and podocyte volume (r = 0.48) and inversely to podocyte number density (r = -0.56), (all significant). F+ podocyte GL3 volume was significantly inversely related to podocyte number density (r = -0.79) and directly to proteinuria. There was no difference in F+ podocyte GL3 volume or volume fraction between age-matched males and females. Thus, in females with Fabry disease GL3 accumulation in F+ podocytes progresses with age in association with podocyte loss and proteinuria, and F+ podocyte GL3 accumulation in females with Fabry is similar to males, consistent with insignificant cross-correction between affected and non-affected podocytes. Hence, these findings have important pathophysiological and clinical implications.

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.

BACKGROUND: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. METHODS: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. RESULTS: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis-up to 93%-of Gb3+ tumor cells in vitro. CONCLUSIONS: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

Clinical relevance of globotriaosylceramide accumulation in Fabry disease and the effect of agalsidase beta in affected tissues.

Fabry disease (FD) is a rare lysosomal storage disorder, characterized by a reduction in α-galactosidase A enzyme activity and the progressive accumulation of globotriaosylceramide (GL3) and its metabolites in the cells of various organs. Agalsidase beta, an enzyme replacement therapy (ERT), is approved for use in patients with FD in Europe, Canada, Australia, South America, and Asia, and is the only ERT approved for use in the United States. In this review, we discuss the clinical relevance of GL3 accumulation, the effect of agalsidase beta on GL3 in target tissues, and the association between treatment-related tissue GL3 clearance and long-term structure, function, or clinical outcomes. Accumulation of GL3 in the kidney, heart, vasculature, neurons, skin, gastrointestinal tract and auditory system correlates to cellular damage and irreversible organ damage, as a result of sclerosis, fibrosis, apoptosis, inflammation, and endothelial dysfunction. Damage leads to renal dysfunction and end-stage renal disease; myocardial hypertrophy with heart failure and arrhythmias; ischemic stroke; neuropathic pain; skin lesions; intestinal ischemia and dysmotility; and hearing loss. Treatment with agalsidase beta is effective in substantially clearing GL3 in a range of cells from the tissues affected by FD. Agalsidase beta has also been shown to slow renal decline and lower the overall risk of clinical progression, demonstrating an indirect link between treatment-related GL3 clearance and stabilization of FD.

Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosome storage disease (LSD) characterized by significantly reduced intracellular autophagy function. This contributes to the progression of intracellular pathologic signaling and can lead to organ injury. Phospholipid-polyethyleneglycol-capped Ceria-Zirconia antioxidant nanoparticles (PEG-CZNPs) have been reported to enhance autophagy flux. We analyzed whether they suppress globotriaosylceramide (Gb3) accumulation by enhancing autophagy flux and thereby attenuate kidney injury in both cellular and animal models of FD. RESULTS: Gb3 was significantly increased in cultured human renal proximal tubular epithelial cells (HK-2) and human podocytes following the siRNA silencing of α galactosidase A (α-GLA). PEG-CZNPs effectively reduced the intracellular accumulation of Gb3 in both cell models of FD and improved both intracellular inflammation and apoptosis in the HK-2 cell model of FD. Moreover these particles attenuated pro fibrotic cytokines in the human podocyte model of FD. This effect was revealed through an improvement of the intracellular autophagy flux function and a reduction in reactive oxygen species (ROS). An FD animal model was generated in which 4-week-old male B6;129-Glatm1Kul/J mice were treated for 8 weeks with 10 mg/kg of PEG-CZNPs (twice weekly via intraperitoneal injection). Gb3 levels were reduced in the kidney tissues of these animals, and their podocyte characteristics and autophagy flux functions were preserved. CONCLUSIONS: PEG-CZNPs alleviate FD associated kidney injury by enhancing autophagy function and thus provide a foundation for the development of new drugs to treat of storage disease.

The binding mechanism of the virulence factor Streptococcus suis adhesin P subtype to globotetraosylceramide is associated with systemic disease.

Streptococcus suis is part of the pig commensal microbiome but strains can also be pathogenic, causing pneumonia and meningitis in pigs as well as zoonotic meningitis. According to genomic analysis, S. suis is divided into asymptomatic carriage, respiratory and systemic strains with distinct genomic signatures. Because the strategies to target pathogenic S. suis are limited, new therapeutic approaches are needed. The virulence factor S. suis adhesin P (SadP) recognizes the galabiose Galα1-4Gal-oligosaccharide. Based on its oligosaccharide fine specificity, SadP can be divided into subtypes PN and PO We show here that subtype PN is distributed in the systemic strains causing meningitis, whereas type PO is found in asymptomatic carriage and respiratory strains. Both types of SadP are shown to predominantly bind to pig lung globotriaosylceramide (Gb3). However, SadP adhesin from systemic subtype PN strains also binds to globotetraosylceramide (Gb4). Mutagenesis studies of the galabiose-binding domain of type PN SadP adhesin showed that the amino acid asparagine 285, which is replaced by an aspartate residue in type PO SadP, was required for binding to Gb4 and, strikingly, was also required for interaction with the glycomimetic inhibitor phenylurea-galabiose. Molecular dynamics simulations provided insight into the role of Asn-285 for Gb4 and phenylurea-galabiose binding, suggesting additional hydrogen bonding to terminal GalNAc of Gb4 and the urea group. Thus, the Asn-285-mediated molecular mechanism of type PN SadP binding to Gb4 could be used to selectively target S. suis in systemic disease without interfering with commensal strains, opening up new avenues for interventional strategies against this pathogen.

Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galß1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galß1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease.

Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(-2) containing the lyso-Gb3(-2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(-2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

An antibody binding-based fluorescent assay for the rapid quantification of globotriaosylceramide levels in human Fabry cells.

Fabry disease is caused by reduced α-GAL A activity and accumulation of globotriaosylceramide (Gb3). Here, we describe a microplate Gb3 assay using fluorophore-tagged antibody and crude cellular lipid extracts. The assay is able to detect higher Gb3 concentrations in human Fabry cells compared to non-diseased cells. This result was verified by immunofluorescence staining that revealed large amounts of Gb3 deposits in Fabry cell lines, demonstrating the accuracy of this method. This assay may provide the basis for detecting Fabry disease by quantifying Gb3 deposits from human biological samples, for example, from urine and blood.

Urinary mulberry bodies as a potential biomarker for early diagnosis and efficacy assessment of enzyme replacement therapy in Fabry nephropathy.

BACKGROUND: The inability of enzyme replacement therapy (ERT) to prevent progression of Fabry nephropathy (FN) in the presence of >1 g/day proteinuria underscores the necessity of identifying effective biomarkers for early diagnosis of FN preceding proteinuria. Here we attempted to identify biomarkers for early detection of FN. METHODS: Fifty-one Fabry disease (FD) patients were enrolled. Urinary mulberry bodies (uMBs) were immunostained for globotriaosylceramide (Gb3) and renal cell markers to determine their origin. The association between semiquantitative uMB excretion and the histological severity of podocyte vacuolation was investigated in seven patients using the vacuolated podocyte:glomerular average area ratio. The association between the semiquantitative estimate of uMB excretion and duration of ERT was analyzed. A longitudinal study was conducted to assess the effect of ERT on uMB excretion. RESULTS: Thirty-two patients (63%) had uMBs, while only 31% showed proteinuria. The uMBs were positive for Gb3, lysosomal-associated membrane protein 1 and podocalyxin, suggesting they were derived from lysosomes with Gb3 accumulation in podocytes. We observed more severe podocyte vacuolation with increased uMB excretion (P = 0.03 for trend); however, the same was not observed with increased proteinuria. The percentage of patients with substantial uMB excretion increased with shorter ERT duration (P = 0.018). Eighteen-month-long ERT reduced uMB excretion (P = 0.03) without affecting proteinuria. CONCLUSIONS: uMB excretion, implying ongoing podocyte injury, preceded proteinuria in most patients. Semiquantitative uMB estimates can serve as novel biomarkers for early FN diagnosis and for monitoring the efficacy of FD-specific therapies.

Pathogenesis and Molecular Mechanisms of Anderson-Fabry Disease and Possible New Molecular Addressed Therapeutic Strategies.

Anderson-Fabry disease (AFD) is a rare disease with an incidenceof approximately 1:117,000 male births. Lysosomal accumulation of globotriaosylceramide (Gb3) is the element characterizing Fabry disease due to a hereditary deficiency α-galactosidase A (GLA) enzyme. The accumulation of Gb3 causes lysosomal dysfunction that compromises cell signaling pathways. Deposition of sphingolipids occurs in the autonomic nervous system, dorsal root ganglia, kidney epithelial cells, vascular system cells, and myocardial cells, resulting in organ failure. This manuscript will review the molecular pathogenetic pathways involved in Anderson-Fabry disease and in its organ damage. Some studies reported that inhibition of mitochondrial function and energy metabolism plays a significant role in AFD cardiomyopathy and in kidney disease of AFD patients. Furthermore, mitochondrial dysfunction has been reported as linked to the dysregulation of the autophagy-lysosomal pathway which inhibits the mechanistic target of rapamycin kinase (mTOR) mediated control of mitochondrial metabolism in AFD cells. Cerebrovascular complications due to AFD are caused by cerebral micro vessel stenosis. These are caused by wall thickening resulting from the intramural accumulation of glycolipids, luminal occlusion or thrombosis. Other pathogenetic mechanisms involved in organ damage linked to Gb3 accumulation are endocytosis and lysosomal degradation of endothelial calcium-activated intermediate-conductance potassium ion channel 3.1 (KCa3.1) via a clathrin-dependent process. This process represents a crucial event in endothelial dysfunction. Several studies have identified the deacylated form of Gb3, globotriaosylsphingosine (Lyso-Gb3), as the main catabolite that increases in plasma and urine in patients with AFD. The mean concentrations of Gb3 in all organs and plasma of Galactosidase A knockout mice were significantly higher than those of wild-type mice. The distributions of Gb3 isoforms vary from organ to organ. Various Gb3 isoforms were observed mainly in the kidneys, and kidney-specific Gb3 isoforms were hydroxylated. Furthermore, the action of Gb3 on the KCa3.1 channel suggests a possible contribution of this interaction to the Fabry disease process, as this channel is expressed in various cells, including endothelial cells, fibroblasts, smooth muscle cells in proliferation, microglia, and lymphocytes. These molecular pathways could be considered a potential therapeutic target to correct the enzyme in addition to the traditional enzyme replacement therapies (ERT) or drug chaperone therapy.

2021 TSOC Expert Consensus on the Clinical Features, Diagnosis, and Clinical Management of Cardiac Manifestations of Fabry Disease.

Fabry disease (FD) is an X-linked, rare inherited lysosomal storage disease caused by α-galactosidase A gene variants resulting in deficient or undetectable α-galactosidase A enzyme activity. Progressive accumulation of pathogenic globotriaosylceramide and its deacylated form globotriaosylsphingosine in multiple cell types and organs is proposed as main pathophysiology of FD, with elicited pro-inflammatory cascade as alternative key pathological process. The clinical manifestations may present with either early onset and multisystemic involvement (cutaneous, neurological, nephrological and the cardiovascular system) with a progressive disease nature in classic phenotype, or present with a later-onset course with predominant cardiac involvement (non-classical or cardiac variant; e.g. IVS4+919G>A in Taiwan) from missense variants. In either form, cardiac involvement is featured by progressive cardiac hypertrophy, myocardial fibrosis, various arrhythmias, and heart failure known as Fabry cardiomyopathy with potential risk of sudden cardiac death. Several plasma biomarkers and advances in imaging modalities along with novel parameters, cardiac magnetic resonance (CMR: native T1/T2 mapping) for myocardial tissue characterization or echocardiographic deformations, have shown promising performance in differentiating from other etiologies of cardiomyopathy and are presumed to be helpful in assessing the extent of cardiac involvement of FD and in guiding or monitoring subsequent treatment. Early recognition from extra-cardiac red flag signs either in classic form or red flags from cardiac manifestations in cardiac variants, and awareness from multispecialty team work remains the cornerstone for timely managements and beneficial responses from therapeutic interventions (e.g. oral chaperone therapy or enzyme replacement therapy) prior to irreversible organ damage. We aim to summarize contemporary knowledge based on literature review and the gap or future perspectives in clinical practice of FD-related cardiomyopathy in an attempt to form a current expert consensus in Taiwan.

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.