Maria Fuller

Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.

Recovery of Airway Cystic Fibrosis Transmembrane Conductance Regulator Function in Mice with Cystic Fibrosis After Single-Dose Lentivirus-Mediated Gene Transfer

The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.

A lipidomic screen of palmitate-treated MIN6 β-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking

Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic β-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 β-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic β-cells.

Newborn Screening for Lysosomal Storage Disorders: Clinical Evaluation of a Two-Tier Strategy

Objective. To evaluate the use of protein markers using immune-quantification assays and of metabolite markers using tandem mass spectrometry for the identification, at birth, of individuals who have a lysosomal storage disorder. Methods. A retrospective analysis was conducted of Guthrie cards that were collected from newborns in Denmark during the period 1982–1997. Patients whose lysosomal storage disorder (LSD; 47 representing 12 disorders) was diagnosed in Denmark during the period 1982–1997 were selected, and their Guthrie cards were retrieved from storage. Control cards (227) were retrieved from the same period. Additional control cards (273) were collected from the South Australian Screening Centre (Australia). Results. From 2 protein and 94 metabolite markers, 15 were selected and evaluated for their use in the identification of LSDs. Glycosphingolipid and oligosaccharide markers showed 100% sensitivity and specificity for the identification of Fabry disease, α-mannosidosis, mucopolysaccharidosis (MPS) IVA, MPS IIIA, Tay-Sachs disease, and I-cell disease. Lower sensitivities were observed for Gaucher disease and sialidosis. No useful markers were identified for Krabbe disease, MPS II, Pompe disease, and Sandhoff disease. The protein markers LAMP-1 and saposin C were not able to differentiate individuals who had an LSD from the control population. Conclusions. Newborn screening for selected LSDs is possible with current technology. However, additional development is required to provide a broad coverage of disorders in a single, viable program.

Early neurodegeneration progresses independently of microglial activation by heparan sulfate in the brain of mucopolysaccharidosis IIIB mice.

Background In mucopolysaccharidosis type IIIB, a lysosomal storage disease causing early onset mental retardation in children, the production of abnormal oligosaccharidic fragments of heparan sulfate is associated with severe neuropathology and chronic brain inflammation. We addressed causative links between the biochemical, pathological and inflammatory disorders in a mouse model of this disease. Methodology/Principal Findings In cell culture, heparan sulfate oligosaccharides activated microglial cells by signaling through the Toll-like receptor 4 and the adaptor protein MyD88. CD11b positive microglial cells and three-fold increased expression of mRNAs coding for the chemokine MIP1α were observed at 10 days in the brain cortex of MPSIIIB mice, but not in MPSIIIB mice deleted for the expression of Toll-like receptor 4 or the adaptor protein MyD88, indicating early priming of microglial cells by heparan sulfate oligosaccharides in the MPSIIIB mouse brain. Whereas the onset of brain inflammation was delayed for several months in doubly mutant versus MPSIIIB mice, the onset of disease markers expression was unchanged, indicating similar progression of the neurodegenerative process in the absence of microglial cell priming by heparan sulfate oligosaccharides. In contrast to younger mice, inflammation in aged MPSIIIB mice was not affected by TLR4/MyD88 deficiency. Conclusions/Significance These results indicate priming of microglia by HS oligosaccharides through the TLR4/MyD88 pathway. Although intrinsic to the disease, this phenomenon is not a major determinant of the neurodegenerative process. Inflammation may still contribute to neurodegeneration in late stages of the disease, albeit independent of TLR4/MyD88. The results support the view that neurodegeneration is primarily cell autonomous in this pediatric disease.

Characterization of a C57BL/6 congenic mouse strain of mucopolysaccharidosis type IIIA

The original mucopolysaccharidosis type IIIA (MPS IIIA) mice were identified in a mixed background with contributions from four different strains. To ensure long-term stability and genetic homogeneity of this lysosomal storage disease (LSD) model, the aim of this study was to develop and characterize a C57BL/6 congenic strain. The B6.Cg-Sgsh(mps3a) strain compares favorably with the original mixed donor strain, exhibiting low liver sulfamidase activity and significant brain heparan sulfate-derived disaccharide elevation from birth. A rapid increase in brain disaccharide levels occurred after birth, with a plateau reached by 13 weeks of age at 110x the levels observed in brains of age-matched unaffected mice. Typical lysosomal inclusions were observed in cerebral cortical and cerebellar neurons and in liver hepatocytes and Kupffer cells. Ubiquitin-positive spheroids and GM(2)-ganglioside were also detected in brain. Using the Morris water maze in male mice, impaired memory and spatial learning was evident at 20 weeks of age in B6.Cg-Sgsh(mps3a) MPS IIIA mice. Other behavioral changes include motor, cognitive and sensory deficits, and aggression. Male B6.Cg-Sgsh(mps3a) MPS IIIA mice exhibited more behavioral abnormalities than B6.Cg-Sgsh(mps3a) MPS IIIA females, as observed previously in the original mixed background strain. Affected mice generally survive to 9 to 12 months of age, before death or euthanasia for humane reasons. Overall, minor differences were apparent between the new congenic and previously described mixed MPS IIIA strains. Availability of an in-bred strain will ensure more reproducible experimental outcomes thereby assisting in our goal of developing effective therapies for LSD with central nervous system disease.

Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra‐cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally‐occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra‐cerebrospinal fluid sulphamidase delivery (100 μg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate‐oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra‐cerebrospinal fluid enzyme. Reductions in micro‐ and astrogliosis and delayed development of ubiquitin‐positive lesions were seen in both groups. A third group of intravenous‐only treated mice did not exhibit clinical or neuropathological improvements. Intra‐cerebrospinal fluid injection of sulphamidase effectively, but dose‐dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.

Disease-Specific Markers for the Mucopolysaccharidoses

Unprecedented demands are now placed on clinicians for early diagnosis as we enter into an era of advancing treatment opportunities for the mucopolysaccharidoses (MPS). Biochemical monitoring of any therapeutic avenue will also be prerequisite. To this end, we aimed to identify a range of urinary oligosaccharides that could be used to identify and characterize patients with MPS. We analyzed 94 urine samples from 68 patients with MPS and 26 control individuals for oligosaccharides derived from glycosaminoglycans using electrospray ionization-tandem mass spectrometry. The oligosaccharide profile for each patient group was compared with that of the control group. The Mann-Whitney U test was used to measure the difference between each patient group and the controls for each analyte. Urine samples from patients before and at successive times after bone marrow transplantation were also evaluated. A number of oligosaccharides were identified in the urine of each MPS subtype, and for each of these, specific oligosaccharide profiles were formulated. These profiles enabled the identification of all 68 patients and their subtypes with the exception of MPS IIIB and IIIC. Selected oligosaccharides were used to assess three individuals after a bone marrow transplant, and, in each case, a substantial reduction in the level of diagnostic oligosaccharides, posttransplantation, was observed. The identification and measurement of glycosaminoglycan-derived oligosaccharides in urine provides a sensitive and specific screen for the early identification of individuals with MPS. The resulting oligosaccharide profiles not only characterize subtype but also provide a disease-specific fingerprint for the biochemical monitoring of current and proposed therapies.

Effect of lysosomal storage on bis(monoacylglycero)phosphate

BMP [bis(monoacylglycero)phosphate] is an acidic phospholipid and a structural isomer of PG (phosphatidylglycerol), consisting of lysophosphatidylglycerol with an additional fatty acid esterified to the glycerol head group. It is thought to be synthesized from PG in the endosomal/lysosomal compartment and is found primarily in multivesicular bodies within the same compartment. In the present study, we investigated the effect of lysosomal storage on BMP in cultured fibroblasts from patients with eight different LSDs (lysosomal storage disorders) and plasma samples from patients with one of 20 LSDs. Using ESI-MS/MS (electrospray ionization tandem MS), we were able to demonstrate either elevations or alterations in the individual species of BMP, but not of PG, in cultured fibroblasts. All affected cell lines, with the exception of Fabry disease, showed a loss of polyunsaturated BMP species relative to mono-unsaturated species, and this correlated with the literature reports of lysosomal dysfunction leading to elevations of glycosphingolipids and cholesterol in affected cells, processes thought to be critical to the pathogenesis of LSDs. Plasma samples from patients with LSDs involving storage in macrophages and/or with hepatomegaly showed an elevation in the plasma concentration of the C(18:1)/C(18:1) species of BMP when compared with control plasmas, whereas disorders involving primarily the central nervous system pathology did not. These results suggest that the release of BMP is cell/tissue-specific and that it may be useful as a biomarker for a subset of LSDs.

Laronidase treatment of mucopolysaccharidosis I.

The lysosomal storage disorder (LSD) mucopolysaccharidosis type I (MPS I, McKusick 25280, Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome) is caused by a deficiency in the lysosomal enzyme, α-L-iduronidase (EC 3.2.1.76). MPS I patients can present within a diverse clinical spectrum, ranging from classical Hurler syndrome to attenuated Scheie syndrome. Laronidase (Aldurazyme®) enzyme replacement therapy has been developed as a treatment strategy for MPS I patients and has been approved for clinical practice. Here we review the pre-clinical studies and clinical trials that have been used to demonstrate that intravenous laronidase therapy is well tolerated and effective for treating MPS I patients who do not have neuronal pathology. Current challenges for a viable treatment strategy for all MPS I patients include development of an early screening protocol that identifies patients before the onset of irreversible pathology, methods to predict disease severity, appropriate treatment for neuropathology, and an effective patient monitoring regimen.