Myelin abnormalities in the optic and sciatic nerves in mice with GM1-gangliosidosis.

GM1-gangliosidosis is a glycosphingolipid lysosomal storage disease involving accumulation of GM1 and its asialo form (GA1) primarily in the brain. Thin-layer chromatography and X-ray diffraction were used to analyze the lipid content/composition and the myelin structure of the optic and sciatic nerves from 7- and 10-month old ß-galactosidase (ß-gal) +/? and ß-gal -/- mice, a model of GM1gangliosidosis. Optic nerve weight was lower in the ß-gal -/- mice than in unaffected ß-gal +/? mice, but no difference was seen in sciatic nerve weight. The levels of GM1 and GA1 were significantly increased in both the optic nerve and sciatic nerve of the ß-gal -/- mice. The content of myelin-enriched cerebrosides, sulfatides, and plasmalogen ethanolamines was significantly lower in optic nerve of ß-gal -/- mice than in ß-gal +/? mice; however, cholesteryl esters were enriched in the ß-gal -/- mice. No major abnormalities in these lipids were detected in the sciatic nerve of the ß-gal -/- mice. The abnormalities in GM1 and myelin lipids in optic nerve of ß-gal -/- mice correlated with a reduction in the relative amount of myelin and periodicity in fresh nerve. By contrast, the relative amount of myelin and periodicity in the sciatic nerves from control and ß-gal -/- mice were indistinguishable, suggesting minimal pathological involvement in sciatic nerve. Our results indicate that the greater neurochemical pathology observed in the optic nerve than in the sciatic nerve of ß-gal -/- mice is likely due to the greater glycolipid storage in optic nerve.

Ganglioside storage diseases: on the road to management.

Although the biochemical and genetic basis for the GM1 and GM2 gangliosidoses has been known for decades, effective therapies for these diseases remain in early stages of development. The difficulty with many therapeutic strategies for treating the gangliosidoses comes largely from their inability to remove stored ganglioside once it accumulates in central nervous system (CNS) neurons and glia. This chapter highlights advances made using substrate reduction therapy and gene therapy in reducing CNS ganglioside storage. Information obtained from mouse and feline models provides insight on therapeutic strategies that could be effective in human clinical trials. In addition, information is presented showing how a calorie-restricted diet might facilitate therapeutic drug delivery to the CNS. The development of multiple new therapeutic approaches offers hope that longer-term management of these diseases can be achieved. It is also clear that multiple therapeutic strategies will likely be needed to provide the most complete management.

Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain.

Filipin is an antibiotic polyene widely used as a histochemical marker for cholesterol. We previously reported cholesterol/filipin-positive staining in brain of ß-galactosidase (ß-gal) knockout ((-/-)) mice (GM1 gangliosidosis). The content and distribution of cholesterol and gangliosides was analyzed in plasma membrane (PM) and microsomal (MS) fractions from whole-brain tissue of 15 week-old control (ß-gal(+/-)) and GM1 gangliosidosis (ß-gal(-/-)) mice. Total ganglioside content (µg sialic acid/mg protein) was 3-fold and 7-fold greater in the PM and MS fractions, respectively, in ßgal(-/-) mice than in ßgal(+/-) mice. GM1 content was 30-fold and 50-fold greater in the PM and MS fractions, respectively. In contrast, unesterified cholesterol content (µg/mg protein) was similar in the PM and the MS fractions of the ßgal(-/-) and ßgal(+/-) mice. Filipin is known to bind to various sterol derivatives and phospholipids on thin-layer chromatograms. Biochemical evidence is presented showing that filipin also binds to GM1 with an affinity similar to that for cholesterol, with a corresponding fluorescent reaction. Our data suggest that the GM1 storage seen in the ß-gal(-/-) mouse contributes to the filipin ultraviolet fluorescence observed in GM1 gangliosidosis brain. The data indicate that in addition to cholesterol, filipin can also be useful for detecting GM1.

Lipid composition of whole brain and cerebellum in Hurler syndrome (MPS IH) mice.

Hurler syndrome (MPS IH) is caused by a mutation in the gene encoding alpha-L-iduronidase (IDUA) and leads to the accumulation of partially degraded glycosaminoglycans (GAGs). Ganglioside content is known to increase secondary to GAG accumulation. Most studies in organisms with MPS IH have focused on changes in gangliosides GM3 and GM2, without the study of other lipids. We evaluated the total lipid distribution in the whole brain and cerebellum of MPS IH (Idua⁻/⁻) and control (Idua(+/?)) mice at 6 months and at 12 months of age. The content of total sialic acid and levels of gangliosides GM3, GM2, and GD3 were greater in the whole brains of Idua⁻/⁻ mice then in Idua (+/?) mice at 12 months of age. No other significant lipid differences were found in either whole brain or in cerebellum at either age. The accumulation of ganglioside GD3 suggests that neurodegeneration occurs in the Idua⁻/⁻) mouse brain, but not to the extent seen in human MPS IH brain.

Restricted ketogenic diet enhances the therapeutic action of N-butyldeoxynojirimycin towards brain GM2 accumulation in adult Sandhoff disease mice.

Sandhoff disease is an autosomal recessive, neurodegenerative disease involving the storage of brain ganglioside GM2 and asialo-GM2. Previous studies showed that caloric restriction, which augments longevity, and N-butyldeoxynojirimycin (NB-DNJ, Miglustat), an imino sugar that hinders the glucosyltransferase catalyzing the first step in glycosphingolipid biosynthesis, both increase longevity and improve motor behavior in the beta-hexosaminidase (Hexb) knockout (-/-) murine model of Sandhoff disease. In this study, we used a restricted ketogenic diet (KD-R) and NB-DNJ to combat ganglioside accumulation. Adult Hexb-/- mice were placed into one of the following groups: (i) a standard diet (SD), (ii) a SD with NB-DNJ (SD + NB-DNJ), (iii) a KD-R, and (iv) a KD-R with NB-DNJ (KD-R + NB-DNJ). Forebrain GM2 content (mug sialic acid/100 mg dry wt) in the four groups was 375 +/- 15, 312 +/- 8, 340 +/- 28, and 279 +/- 26, respectively, indicating an additive interaction between NB-DNJ and the KD-R. Most interestingly, brain NB-DNJ content was 3.5-fold greater in the KD-R + NB-DNJ mice than in the SD + NB-DNJ mice. These data suggest that the KD-R and NB-DNJ may be a potential combinatorial therapy for Sandhoff disease by enhancing NB-DNJ delivery to the brain and may allow lower dosing to achieve the same degree of efficacy as high dose monotherapy.

Brain lipid analysis in mice with Rett syndrome.

Rett syndrome (RS) is an X-linked neurodevelopmental disorder mostly involving mutations in the gene for methyl-CpG-binding protein 2 (MECP2). Ganglioside abnormalities were previously found in cerebrum and cerebellum in RS patients. We evaluated total lipid distribution in cerebrum/brainstem, hippocampus, and cerebellum in male mice carrying either the Mecp2 (tm1.1Bird) knockout mutation or the Mecp2 (308/y) deletion mutation. The concentration of the neuronal enriched ganglioside GD1a was significantly lower in the cerebrum/brainstem of Mecp2 (tm1.1Bird) mice than in that of age matched controls, but was not reduced in the Mecp2 (308/y) mice. No other differences in brain lipid content, including myelin-enriched cerebrosides, were detected in mice with either type of Mecp2 mutation. These findings indicate that the poor motor performance previously reported in the RS mutant mice is not associated with major brain lipid abnormalities and that most previous brain lipid abnormalities observed in RS patients were not observed in the Mecp2 (tm1.1Bird) or the Mecp2 (308/y) RS mice.

Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.

Sphingosine-1-phosphate is a sphingolipid metabolite that regulates cell proliferation, migration and apoptosis through specific signaling pathways. Sphingosine-1-phosphate lyase catalyzes the conversion of sphingosine-1-phosphate to ethanolamine phosphate and a fatty aldehyde. We report the cloning of the Drosophila sphingosine-1-phosphate lyase gene (Sply) and demonstrate its importance for adult muscle development and integrity, reproduction and larval viability. Sply expression is temporally regulated, with onset of expression during mid-embryogenesis. Sply null mutants accumulate both phosphorylated and unphosphorylated sphingoid bases and exhibit semi-lethality, increased apoptosis in developing embryos, diminished egg-laying, and gross pattern abnormalities in dorsal longitudinal flight muscles. These defects are corrected by restoring Sply expression or by introduction of a suppressor mutation that diminishes sphingolipid synthesis and accumulation of sphingolipid intermediates. This is the first demonstration of novel and complex developmental pathologies directly linked to a disruption of sphingolipid catabolism in metazoans.

Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans.

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.