Autosomal recessive mutations in the galactosidase beta 1 (GLB1) gene cause lysosomal βgalactosidase (Beta-Gal) deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human Beta-Gal (rhBeta-Gal) produced in Chinese hamster ovary cells enabled direct and precise rhBeta-Gal delivery to acidified lysosomes. A single, low dose (3 nM) of rhBeta-Gal was sufficient for normalizing Beta-Gal activity and mediating substrate clearance for several weeks. We found that rhBeta-Gal uptake by the fibroblasts is dose dependent and saturable, and can be competitively inhibited by mannose-6-phophate, suggesting cation-independent, mannose-6phophate receptor-mediated endocytosis from the cell-surface. A single intracerebroventricular (ICV)-administered dose of rhBeta-Gal (100 micrograms) resulted in broad bilateral ____________________________ GM1 gangliosidosis exhibits an autosomal recessive mode of inheritance and arises from http://www.jbc.org/cgi/

IntroductionGM1 gangliosidosis is a rare autosomal recessive genetic disorder caused by the disruption of the GLB1 gene that encodes β-galactosidase, a lysosomal hydrolase that removes β-linked galactose from the non-reducing end of glycans. Deficiency of this catabolic enzyme leads to the lysosomal accumulation of GM1 and its asialo derivative GA1 in β-galactosidase deficient patients and animal models. In addition to GM1 and GA1, there are other glycoconjugates that contain β-linked galactose whose metabolites are substrates for β-galactosidase. For example, a number of N-linked glycan structures that have galactose at their non-reducing end have been shown to accumulate in GM1 gangliosidosis patient tissues and biological fluids.ObjectiveIn this study, we attempt to fully characterize the broad array of GLB1 substrates that require GLB1 for their lysosomal turnover.ResultsUsing tandem mass spectrometry and glycan reductive isotope labeling with data-dependent mass spectrometry, we have confirmed the accumulation of glycolipids (GM1 and GA1) and N-linked glycans with terminal beta-linked galactose. We have also discovered a novel set of core 1 and 2 O-linked glycan metabolites, many of which are part of structurally-related isobaric series that accumulate in disease. In the brain of GLB1 null mice, the levels of these glycan metabolites increased along with those of both GM1 and GA1 as a function of age. In addition to brain tissue, we found elevated levels of both N-linked and O-linked glycan metabolites in a number of peripheral tissues and in urine. Both brain and urine samples from human GM1 gangliosidosis patients exhibited large increases in steady state levels for the same glycan metabolites, demonstrating their correlation with this disease in humans as well.ConclusionsOur studies illustrate that GLB1 deficiency is not purely a ganglioside accumulation disorder, but instead a broad oligosaccharidosis that include representatives of many β-linked galactose containing glycans and glycoconjugates including glycolipids, N-linked glycans, and various O-linked glycans. Accounting for all β-galactosidase substrates that accumulate when this enzyme is deficient increases our understanding of this severe disorder by identifying metabolites that may drive certain aspects of the disease and may also serve as informative disease biomarkers to fully evaluate the efficacy of future therapies.

Title we quantified the effects of individual BDE congeners on not only dendritic but also axonal 36 growth since both are regulated by RyR-dependent mechanisms, and both are critical 37 determinants of neuronal connectivity. Primary neuronal-glial co-cultures dissociated from the 38 neonatal rat hippocampus were exposed to BDE-47 or BDE-49 in the culture medium at 39 concentrations ranging from 20 pM to 2 µM. At these concentrations, neither PBDE congener 40 altered dendritic arborization. In contrast, at concentrations ≥ 200 pM, both congeners delayed 41 neuronal polarization resulting in significant inhibition of axonal outgrowth during the first few 42 days in vitro. The axon inhibitory effects of these PBDE congeners occurred independent of 43 cytotoxicity, and were blocked by pharmacological antagonism of RyR or siRNA knockdown of 44RyR2. These results demonstrate that the molecular and cellular mechanisms by which PBDEs 45 interfere with neurodevelopment overlap with but are distinct from those of NDL PCBs, and 46 suggest that altered patterns of neuronal connectivity may contribute to the developmental 47 neurotoxicity of PBDEs. 48 49

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β-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing β-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by β-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders.