A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease.

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.

Acid sphingomyelinase deficiency. Phenotype variability with prevalence of intermediate phenotype in a series of twenty-five Czech and Slovak patients. A multi-approach study.

A multi-approach study in a series of 25 Czech and Slovak patients with acid sphingomyelinase deficiency revealed a broad phenotypic variability within Niemann-Pick disease types A and B. The clinical manifestation of only 9 patients fulfilled the historical classification: 5 with the rapidly progressive neurovisceral infantile type A and 4 with a slowly progressive visceral type B. Sixteen patients (64%) represented a hitherto scarcely documented 'intermediate type' (IT). Twelve patients showed a protracted neurovisceral course with overt or mild neurological symptoms, three a rapidly progressing fatal visceral affection with rudimentary neurological lesion. One patient died early from a severe visceral disease. The genotype in our patients was represented by 4 frameshift and 14 missense mutations. Six were novel (G166R, R228H, A241V, D251E, D278A, A595fsX601). The Q292K mutation (homoallelic, heteroallelic) was strongly associated with a protracted neurovisceral phenotype (10 of 12 cases). The sphingomyelin loading test in living fibroblasts resulted in total degradation from less than 2% in classical type A to 70-80% in classical type B. In the IT group it ranged from 5% to 49% in a 24 h chase. The liver storage showed three patterns: diffuse, zonal (centrolobular), and discrete submicroscopic. Our series showed a notable variability in both the neurological and visceral lesions as well as in their proportionality and synchrony, and demonstrates a continuum between the historical 'A' and 'B' phenotypes of ASM deficiency. This points to a broad phenotypic potential of ASM deficiency, suggesting the existence of still unknown factors independently controlling the storage level in the visceral and neuronal compartments. This report highlights the important position of the IT in the ASM deficiency phenotype classification. We define IT as a cluster of variants combining clinical features of both the classical types. The protracted neuronopathic variant with overt, borderline or subclinical neurology prevails and is important in view of future enzyme replacement therapy. It appears more common in central Europe. The visceral, rapidly progressing early fatal type has been recognized rarely so far.

Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease.

Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide beta-glucosidase, resulting in the accumulation of glucosylceramide in macrophage-derived cells known as Gaucher cells. Much of the storage material is thought to originate from the turnover of cell membranes, such as phagocytosed red and white blood cells. In this study, an in vitro model of Gaucher disease was developed by treating the murine macrophage cell line J774 with a specific inhibitor of glucosylceramide beta-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state. It was found in this model system that glucosylceramide beta-glucosidase activity could be reduced to about 11-15% of the normal control level before increased storage of glucosylceramide occurred. This in vitro system allows insight into the correlation between enzyme activity and lipid storage as predicted by the theory of residual enzyme activity that was proposed by Conzelmann and Sandhoff.

Toxicity of glucosylsphingosine (glucopsychosine) to cultured neuronal cells: a model system for assessing neuronal damage in Gaucher disease type 2 and 3.

Patients with Gaucher disease have been classified as type 1 nonneuronopathic, type 2 acute neuronopathic, and type 3 chronic neuronopathic phenotypes. Increased quantities of glucocerebroside and glucosylsphingosine (glucopsychosine) are present in the brain of type 2 and type 3 Gaucher patients. Galactosylsphingosine has previously been shown to be neurotoxic in globoid cell leukodystrophy (Krabbe disease). To determine whether glucosylsphingosine is also neurotoxic, we examined its effect on cultured cholinergic neuron-like LA-N-2 cells. When these cells were exposed to 1, 5, or 10 microM glucosylsphingosine for a period of 18 h, they became shriveled, neurite outgrowth was suppressed, and the activities of the lysosomal enzymes glucocerebrosidase, sphingomyelinase, and beta-galactosidase were reduced in a dose-dependent manner. Acetylcholine in cells exposed to glucosylsphingosine also declined. Cells switched to glucosylsphingosine-free medium partially recovered. The data suggest that accumulation of glucosylsphingosine contributes to neuronal dysfunction and destruction in patients with neuronopathic Gaucher disease.

Lipid-protein interactions and effect of local anesthetics in acetylcholine receptor-rich membranes from Torpedo marmorata electric organ.

The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine >> tetracaine approximately procaine.

Reduced rates of axonal and dendritic growth in embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease.

Sandhoff disease is a lysosomal storage disease in which ganglioside GM2 accumulates because of a defective beta-subunit of beta-hexosaminidase. This disease is characterized by neurological manifestations, although the pathogenic mechanisms leading from GM2 accumulation to neuropathology are largely unknown. We now examine the viability, development and rates of neurite growth of embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease, the Hexb-/- mouse. GM2 was detected by metabolic labelling at low levels in wild type (Hexb+/+) neurones, and increased by approximately three-fold in Hexb-/- neurones. Hexb-/- hippocampal neurones were as viable as their wild type counterparts and, moreover, their developmental programme was unaltered because the formation of axons and of the minor processes which eventually become dendrites was similar in Hexb-/- and Hexb+/+ neurones. In contrast, once formed, a striking difference in the rate of axonal and minor process growth was observed, with changes becoming apparent after 3 days in culture and highly significant after 5 days in culture. Analysis of various parameters of axonal growth suggested that a key reason for the decreased rate of axonal growth was because of a decrease in the formation of collateral axonal branches, the major mechanism by which hippocampal axons elongate in culture. Thus, although the developmental programme with respect to axon and minor process formation and the viability of hippocampal neurones are unaltered, a significant decrease occurs in the rate of axonal and minor process growth in Hexb-/- neurones. These results appear to be in contrast to dorsal root ganglion neurones cultured from 1-month-old Sandhoff mice, in which cell survival is impaired but normal outgrowth of neurones occurs. The possible reasons for these differences are discussed.

Long endogenous dsRNAs can induce complete gene silencing in mammalian cells and primary cultures.

Recently, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has rapidly developed to a powerful instrument for specific silencing of gene expression in several organisms, including Caenorhabditis elegans, Drosophila melanogaster, and plants. The finding that synthetic small interfering RNAs (siRNAs) of 21 nt as well as stable, endogenously expressed, large dsRNA are suited to specifically induce gene silencing in mammalian cells offered the possibility of expanding this technique to mammalian systems. In this work, we engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes. We used this technique to specifically silence genes coding for glucosylceramide synthase (GCS), the sphingolipid activator protein precursor (SAP), and glucocerebrosidase (GBA), all implicated in glycosphingolipid metabolism. From a 1600-bp inverted repeat DNA template, a dsRNA of 800 bp is expressed and predicted to mediate the specific suppression of the corresponding gene by RNAi. Remarkably, we were able to use this method to achieve complete inhibition of those genes we targeted in different cultured human cell lists. These findings testify to the generality of RNAi application in suppressing gene expression in mammalian cells.

Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

Uptake of mannose-terminal glucocerebrosidase in cultured human cholinergic and dopaminergic neuron cell lines.

Enzyme replacement therapy has been shown to be particularly effective for patients with type 1 (non-neuronopathic) Gaucher disease. However, intravenously administered glucocerebrosidase does not reverse or halt the progression of brain damage in patients with type 2 (acute neuronopathic) Gaucher disease. A previous investigation revealed that intracerebral infusion of mannose-terminal glucocerebrosidase was safe in experimental animals. The enzyme had a comparatively long half-life in the brain. It was transported by convection from the site of infusion along white matter fiber tracts to the cerebral cortex where it was endocytosed by neurons. In anticipation of intracerebral administration of mannose-terminal glucocerebrosidase to patients with type 2 Gaucher disease, it was important to learn the mechanism involved in its cellular uptake. We therefore compared the endocytosis of this enzyme by J774 macrophage cells with that in two human neuronal cell lines and a human astrocyte cell line. Mannose-terminal glucocerebrosidase was taken up by cholinergic LA-N-2 cells, but to a much lower extent than by macrophages. Considerably less of the enzyme was endocytosed by dopaminergic SH-SY5Y cells. It was not taken up by NHA astrocytes. The findings provide encouragement for an exploration of intracerebral administration of glucocerebrosidase in patients with type 2 Gaucher disease.

Complete analysis of the glycosylation and disulfide bond pattern of human beta-hexosaminidase B by MALDI-MS.

beta-hexosaminidase B is an enzyme that is involved in the degradation of glycolipids and glycans in the lysosome. Mutation in the HEXB gene lead to Sandhoff disease, a glycolipid storage disorder characterized by severe neurodegeneration. So far, little structural information on the protein is available. Here, the complete analysis of the disulfide bond pattern of the protein is described for the first time. Additionally, the structures of the N-glycans are analyzed for the native human protein and for recombinant protein expressed in SF21 cells. For the analysis of the disulfide bond structure, the protein was proteolytically digested and the resulting peptides were analyzed by MALDI-MS. The analysis revealed three disulfide bonds (C91-C137; C309-C360; C534-C551) and a free cysteine (C487). The analysis of the N-glycosylation was performed by tryptic digestion of the protein, isolation of glycopeptides by lectin chromatography and mass measurement before and after enzymatic deglycosylation. Carbohydrate structures were calculated from the mass difference between glycosylated and deglycosylated peptide. For beta-hexosaminidase B from human placenta, four N-glycans were identified and analyzed, whereas the recombinant protein expressed in SF21 cells carried only three glycans. In both cases the glycosylation belongs to the mannose-core- or high-mannose-type, and some carbohydrate structures are fucosylated.

Sphingolipid activator proteins: proteins with complex functions in lipid degradation and skin biogenesis.

Sphingolipid activator proteins (SAPs or saposins) are essential cofactors for the lysosomal degradation of membrane-anchored sphingolipids. Four of the five known proteins of this class, SAPs A--D, derive from a single precursor protein and show high homology, whereas the fifth protein, GM2AP, is larger and displays a different secondary structure. Although the main function of all five proteins is assumed to lie in the activation of lipid degradation, their specificities and modes of action seem to differ considerably. It has recently been demonstrated that the action of the proteins is highly enhanced by the presence of acidic lipids in the target membranes. These results have some interesting implications for the topology of lysosomal degradation of lipids and may provide new insights into the function of these interesting proteins, which are ubiquitously expressed in the different tissues of the body. Recent studies indicated that the SAPs play an important role in the biogenesis of the epidermal water barrier, which has been demonstrated by the analysis of the skin phenotype displayed by SAP-knockout mice. The results obtained so far have led to some new insights into the formation of the epidermal water permeability barrier and may lead to a better understanding of this complex process.

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.

Stimulation of acid sphingomyelinase activity by lysosomal lipids and sphingolipid activator proteins.

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substrate-carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent-free conditions only the sphingolipid activator protein SAP-C had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sphingomyelin degradation.

Degradation of membrane-bound ganglioside GM2 by beta -hexosaminidase A. Stimulation by GM2 activator protein and lysosomal lipids.

According to a recent hypothesis, glycosphingolipids originating from the plasma membrane are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures. Since most previous in vitro investigations used micellar ganglioside GM2 as substrate, we studied the degradation of membrane-bound ganglioside GM2 by water-soluble beta-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic lipids such as the lysosomal components bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the degradation of GM2 by beta-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degradation rate of GM2 incorporated into liposomes composed of neutral lysosomal lipids such as dolichol, cholesterol, or phosphatidylcholine was significantly lower than in negatively charged liposomes. This demonstrates that both, the GM2 activator protein and anionic lysosomal phospholipids, are needed to achieve a significant degradation of membrane-bound GM2 under physiological conditions. The interaction of GM2 activator protein with immobilized membranes was studied with surface plasmon resonance spectroscopy at an acidic pH value as it occurs in the lysosomes. Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a significant drop of the resonance signal in the presence of GM2 activator protein. This suggests that in the presence of bis(monoacylglycero)phosphate, which has been shown to occur in inner membranes of the acidic compartment, GM2 activator protein is able to solubilize lipids from the surface of immobilized membrane structures.

Molecular analysis of acid ceramidase deficiency in patients with Farber disease.

Farber disease is a rare, autosomal recessively inherited sphingolipid storage disorder due to the deficient activity of lysosomal acid ceramidase, leading to the accumulation of ceramide in cells and tissues. Here we report the identification of six novel mutations in the acid ceramidase gene causing Farber disease: three point mutations resulting in single amino acid substitutions, one intronic splice site mutation resulting in exon skipping, and two point mutations also leading to occasional or complete exon skipping. Of interest, these latter two mutations occurred in adjacent nucleotides and led to abnormal splicing of the same exon. Expression of the mutated acid ceramidase cDNAs in COS-1 cells and subsequent determination of acid ceramidase residual enzyme activity demonstrated that each of these mutations was the direct cause of the acid ceramidase deficiency in the respective patients. In contrast, two known polymorphisms had no effect on acid ceramidase activity. Metabolic labeling studies in fibroblasts of four patients showed that even though acid ceramidase precursor protein was synthesized in these individuals, rapid proteolysis of the mutated, mature acid ceramidase occurred within the lysosome.

CD95 signaling via ceramide-rich membrane rafts.

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures.

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.

Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.