Recent advances in the biochemistry and genetics of sphingolipidoses

Brain & Development 26 (2004) 497–505 www.elsevier.com/locate/braindev

Review article

Recent advances in the biochemistry and genetics of sphingolipidoses ¨ zkara* Hatice Asuman O Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey Received 11 June 2003; received in revised form 8 January 2004; accepted 24 January 2004

Abstract Sphingolipidoses are a subgroup of lysosomal storage diseases. They are defined as disorders caused by a genetic defect in catabolism of sphingosine-containing lipids. Catabolism of these lipids involves enzymes and activator proteins. After the discovery of lysosomes by de Duve and the demonstration of the first defective lysosomal enzyme by Hers in 1963, the first enzyme deficiency for sphingolipidoses was characterized in 1965 and all the defective enzymes were demonstrated in the last three decades. In 1984, the first activator protein was found and it expanded the concept of sphingolipidoses. In the following years, many researches have been undertaken to understand the molecular basis of these diseases, the mechanism of pathogenesis, the mechanism of lysosomal digestion of glycosphingolipids (GSLs) and the functional domains of lysosomal enzymes. New hypotheses and theories have been put forward for the mechanism of lysosomal digestion and pathogenesis. However, although much has been done, the pathogenesis of sphingolipidoses has not been fully elucidated. Mouse models of these diseases have facilitated the elucidation of pathogenesis and the development of therapeutic strategies for these diseases, which are not treatable at present except for Fabry and type 1 Gaucher disease. The purpose of this review is to collect information on the recent researches related to sphingolipidoses. The review includes the hydrolysis of GSLs in lysosome, mechanism of hydrolysis, pathogenesis and genetics of sphingolipidoses, a brief mouse model and therapeutic strategies of these diseases. q 2004 Elsevier B.V. All rights reserved. Keywords: Sphingolipidoses; Biochemistry; Genetics

1. Introduction Glycosphingolipids (GSLs) are part of the important components of eukaryotic cell membranes. They anchor with their hydrophobic ceramide moieties to the outer leaflet of the cell membrane following the synthesis and posttranslational modifications in endoplasmic reticulum (ER) and in the Golgi apparatus. After their synthesis is completed in the Golgi, GSLs are delivered to the plasma membrane where they are organised into lipid-based microdomains called rafts and caveolae. These lipid microdomains are thought to function as signaling platforms by virtue of their affinity for signaling proteins-transmembrane receptors, GPI-anchored proteins [1,2]. The type and amount of the GSLs may vary according to the type and developmental stage of the cell. The sialic acid containing type of GSLs, gangliosides, form 6% of the membrane lipids particularly in brain cells. The physiological functions of the GSLs are known to be: (a) receptors for endogenous * Fax: þ90-312-310-0580. ¨ zkara). E-mail address: [email protected] (H.A. O 0387-7604/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2004.01.005

and exogenous molecules; (b) a modulator for cell differentiation; and (c) important for the control of cell growth and oncogenic transformation [3]. Characterization of the diseases related to GSL catabolism has revealed the importance of GSL metabolism. The catabolic enzymes and their mechanisms have been understood thoroughly by the help of researches on these disorders. GSL storage diseases are fatal disorders and significant for some populations. It is observed that GSL is stored in most of the lysosomal storage disorders. The percentage for each living birth is 1/7700 [4]. Table 1 and Fig. 1 show the significant GSL storage diseases and related defective enzymes. There is no disease that is defined by the lack of enzyme in the GSL biosynthesis. Probably this type of fetus could not be compatible with life and aborted spontaneously at an early stage of gestation. In the last two decades, new information and theories have been put forward on the (a) enzymology of lysosomal enzymes, (b) the mechanism of the occurrence of clinical heterogeneity, and (c) how lysosomal enzymes could hydrolyze the accumulated GSLs without destroying the lysosomal membrane.

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(2) how intralysosomal multivesicular storage particles exist in the fibroblasts of patients with sphingolipidoses.

Table 1 Summary of major sphingolipidoses Disease

Defective enzyme

Substrate

GM1 gangliosidosis

b-Galactosidase

GM1 ganglioside

Hexosaminidase A Hexosaminidase A and B GM2 ganglioside

GM2 ganglioside Globoside, GM2 ganglioside GM2 ganglioside

a-Galactosidase A Glucocerebrosidase Acid ceramidase

Globotriaosylceramide Glucosylceramide Ceramide

GM2 gangliosidoses (a) Tay-Sachs disease (b) Sandhoff disease (c) Activator deficiency (AB variant) Fabry disease Gaucher disease Farber disease

Metachromatic leukodystrophy (a) Enzyme deficient Arylsulphatase A form (b) Activator deficient Sulfatide activator form Krabbe disease Niemann-Pick disease

Galactosylceramidase Sphingomyelinase

Sulfatide Sulfatide Galactosylceramide Sphingomyelin

By using Southern blot, cloning, polymerase chain reaction (PCR) and PCR-based techniques, gene abnormalities have been determined and the structure of enzyme proteins and their functional sites have been understood. In this review, information obtained from current studies on biochemistry and genetics of sphingolipidoses will be presented.

2. Hydrolysis of GSLs in lysosomes It is necessary to know the degradation mechanism of GSLs in lysosomes in order to understand the pathogenesis of sphingolipidoses. In normal cells, the synthesis and breakdown of GSLs are in balance. GSLs that are to be degraded are endocytosed to lysosomes from the membrane and hydrolyzed there. There are two models that explain the cell degradation of GSLs in the lysosome. In the first model, GSLs in the plasma membrane are budded to the intracellular vesicles as coated pits that can fuse with the early endosomes. Early endosomes combine with lysosomal membranes and the degradation of GSLs occurs within the lysosomal membranes without impairing their integrity by lysosomal enzymes. However, it is not clear how lysosomal enzymes recognize those lipids through the protective glycocalix layer [5,6]. In the second model, budded plasma membranes fuse with the endosomal membrane and form intraendosomal vesicles. The endosome fusion with the lysosomal membrane bears vesicles into the lysosome and form intralysosomal vesicles. Thus, the outer part of the plasma membrane takes part in the outer leaflet of the intralysosomal vesicle in the lysosomes. This model can answer the following questions: (1) how GSLs are hydrolyzed despite the protective glycocalix layer and

3. Mechanism of hydrolysis of GSLs by lysosomal enzymes GSLs are hydrolyzed by specific lysosomal enzymes. Those enzymes cleave the carbohydrate moiety from the non-reduced end of GSLs. The number of monosaccharide moiety in the GSLs is important for their hydrolysis by enzyme or both enzyme and its activator protein. GSLs that contain more than four units of glucose in the carbohydrate chain need only enzymes for hydrolysis. GSLs with shorter carbohydrate chain need not only enzymes but also a second molecule. Those molecules in the non-enzymatic protein structure are called sphingolipid activator proteins (SAPs). The first activator protein SAP-B was defined by Mehl and Jatzkewitz [7] in 1964, for arylsulphatase A activity in sulphatide hydrolysis. Conzelmann and Sandhoff [8] defined the second activator protein called GM2 activator protein which is needed for hexosaminidase A in GM2 ganglioside hydrolysis in 1979. It is located on the fifth chromosome. It contains four exons and three introns. In the following years, in 1988 and 1989, SAP-A, SAP-C, and SAP-D were also characterized [9 – 11]. It is shown that SAPs A – D, called saposin, is synthesized from a single polypeptide by a proteolytic cleavage of precursor protein. SAP precursor is found on the 10th chromosome. It contains 15 exons and 14 introns. GM2 activator and SAP-B bind to the carbohydrate chain of GSLs and serve it to the lysosomal enzyme. SAP-B is the least specific among the activator proteins. It stimulates the hydrolysis of 20 glycolipids by using different enzymes. In vitro SAP-A stimulates glucosylceramidase and galactosylceramidase in the presence of detergent. SAP-C is a membrane active protein that solubilizes lipids. In vivo, fundamentally it is responsible for the degradation of glucosylceramides. SAP-D is necessary for degradation of ceramide with acid ceramidase in vivo [12]. Selective degradation of GSLs by lysosomal enzymes is not yet clear. Three factors are regarded as significant for the selective degradation process by limited and insufficient in vitro studies [13]. These factors are: lateral pressure, lipid composition, and membrane curvature. 3.1. Lateral pressure GM2 activator protein is activated in the membrane when the lateral pressure of the monolayer is lower than 25 mN/M. The lateral pressures of the biologic membranes are higher than this value. 3.2. Lipid composition The membrane of lysosome contains negatively charged lipids. Bis(monoacylglycero)phosphate (BMP), one of those

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Fig. 1. Chemical and metabolic relationships among major glycosphingolipids and the location of genetic blocks. Glu, glucose; Gal, galactose; NANA, N-acetyl neuraminic acid; NAGA, N-acetylgalactosamine.

lipids, is found only in lysosomes and endosomes. Lysosome also contains dolicholphosphate and phosphatidylinositol. It has been experimentally demonstrated that negatively charged lipids play an important role in GSL hydrolysis [14].

3.3. Membrane curvature According to the second topological model of lysosomal degradation, the diameter of intralysosomal vesicle affects the degradation of plasma membrane-derived lipids.

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In vivo, the diameter of the vesicle that exists in the lysosome has been found to range between 50 and 100 nm [15].

4. Pathogenesis of sphingolipidoses Sphingolipidoses can be classified as infantile, juvenile, and adult types according to the onset of the disease and the level of enzymes. Among those types, the infantile type has the worst prognosis. In juvenile and adult types, residual enzyme activity allows slow down of the symptoms of the disease and extends the life span. There is not much information about the pathogenesis of these diseases. However, there are several significant factors that particularly explain the pathogenesis: the biochemical pathway that cells choose in the synthesis of GSLs, differences in GSL types in different cells, toxic compounds derived from stored GSLs and diversity in genotype. 4.1. Biochemical pathway that cells choose in the synthesis of GSLs Two types of synthesis have been defined for GSL’s in cells: de novo, and salvage pathway [16 – 18]. The cells that divide and differentiate prefer de novo synthesis, whereas the differentiated cells that do not divide prefer the salvage pathway. MLD and GM2 gangliosidoses examples can explain the significance of this preferred way on the pathogenesis of sphingolipidoses. Sulphatide catabolism is defective in MLD. If the de novo synthesis is dominant, the patients with MLD will not experience difficulty in myelin synthesis. According to the above-mentioned hypothesis, in a developed nervous system, the formation of sulphatide changes de novo synthesis into the salvage pathway and an enzyme defect affects the formation of myelin. Since sulphatide and galactosylceramide function in the process of sorting of vesicles and transportation of myelin proteins, the maintenance of myelin and transportation of other myelin proteins will be negatively affected in the absence of those substances [19 – 21]. 4.2. Different GSLs in different cells Different GSLs are synthesized in different cell types. Due to these differences, the effects of enzyme defect will vary according to the cell type. For example, GM2 gangliosides that cannot be degraded due to the defective hexosaminidase A are mainly stored in the neurons and affected patients show neurological symptoms [22]. 4.3. Toxic compounds derived from stored GSLs Some sphingolipids that are stored in the membrane because of degrading enzyme deficiency are not toxic for the cell. However, if those substances are stored in large amounts, they interfere with intracellular transport and with

other cell activities. Some GSLs are converted to toxic compounds. Such toxic compounds derived from stored GSLs are indicated in Gaucher, Krabbe, Tay-Sachs and Sandhoff diseases. These are psychosine derivatives in Gaucher and Krabbe diseases, and lysoganglioside GM2 in Tay-Sachs and Sandhoff diseases. These substances are toxic and show lytic properties in cell culture [23 – 26]. It is indicated in a study that lysosphingolipids are potent inhibitors of protein kinase C and interfere with signal transduction in nerve cells [27]. Recent studies also show that sphingolipids are significant molecules that have a role in the intra- and/or intercellular signal transduction. Ceramide, sphingosine, and sphingosine-1-phosphate are defined as second messengers, the latter also act as an extracellular ligand for the endothelial differentiation gene-1 G-protein coupled receptor family [28]. While ceramide and sphingosine-1-phosphate trigger the cascade that result in apoptosis, generation of sphingosine allows the cell to survive. Bioaccumulation of different GSLs and decreased concentration of signaling molecules by enzyme defects may affect signal transduction and may cause different cell response, leading to various clinical symptoms and diversity in pathogenesis [18,29]. 4.4. Genotype differences Genotype differences generally determine the pathogenesis of the disease according to its effect on the enzyme activity level. However, in some particular cases, the same genotype causes formation of different phenotypes. For example, a 12 bp deletion mutation in exon 10 of hexosaminidase a-subunit gene, which is seen in the Turkish population, causes both the infantile type of TaySachs disease and its B1 variant in different patients [30 –32]. Similarly, a mutation in arylsulphatase A gene causes the formation of different phenotypes [33]. When we consider the pathogenesis of these diseases according to the residual enzyme activity level a critical question is raised: Why does a certain amount of enzyme activity lead to a better and longer life in this disease? To answer this question, we need to remember Conzelmann and Sandhoff’s theory [34] (critical threshold theory) on residual enzyme activity which helps to clarify the pathogenesis of heterozygotes, juvenile and adult types of the sphingolipidoses. According to this theory, under normal physiological conditions, lysosomal enzymes work with substrate concentrations below their Km values. In the case of low enzyme concentrations, the enzyme can tolerate the increase of the substrate concentration up to the steady-state concentration. At higher substrate concentrations, the substrate starts to accumulate and clinical findings appear. This explains why the clinical findings of juvenile and adult types of sphingolipidoses appear later than the infantile type and the normal life of heterozygotes. A 10–20% of residual enzyme activity will be sufficient for a normal life in heterozygotes.

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5. Genetics of sphingolipidoses

6. Animal models

The genes of enzymes that are responsible for GSL catabolism have been cloned. Neufeld [35] had classified the findings of these studies under three categories according to the properties of these genes, and this classification has not changed since that time:

Sphingolipidoses that occur naturally in animals (Krabbe disease) and transgenic mouse models help us to understand the pathogenesis of sphingolipidoses and to develop new therapeutic strategies. Mouse models have been created for Tay-Sachs and Sandhoff diseases, GM2 activator deficiency, GM1 gangliosidosis, Fabry, Niemann-Pick type A, Gaucher type II, and galactosialidosis. Only the knockout mice model of Tay-Sachs disease did not develop the disease. The reason for this is that the sialidase enzyme in mice has different properties from the sialidase enzyme in humans. This enzyme can transform GM2 to GA2, which can be hydrolyzed by hexosaminidase B [43]. The accumulation of GM2 in mice, though in small amounts, continues. However, this accumulation is not rapid enough to cause pathologic results [13]. Other models expose similar human diseases. Those models have been summarized in the 2001 edition of The Metabolic and Molecular Bases of Inherited Disease [44 –51].

(1) These enzymes show a sequence homology with the same enzyme genes of other living organisms and the enzymes that catalyze similar reactions. Neufeld had summarized these genes. (2) Promoter regions for b-subunit of b-hexosaminidase and arylsulphatase A include TATA box and GC rich SP1 binding site [35]. In a-galactosidase gene SP1 region is within the TATA box [36,37]. The b-hexosaminidase a-subunit gene does not include TATA box and glycocerebrosidase gene does not have SP1 region. The differences in the promoter regions cause a different expression of reporter gene in different cell types [38,39]. (3) Proteins that alternate splicing products have been produced for some genes like sphingomyelinase gene groups. However, it is denoted that they are not active enzymes and they cannot function in catalysis. The physiological functions of those alternate transcripts are completely known [40 –42]. Cloning the genes, the wide use of PCR and related technologies since 1989 have enabled sphingolipidoses causing mutations to be determined (Table 2). The results of mutation analysis showed that: (1) mutations that cause sphingolipidoses are heterogeneous; (2) a correlation is possible between genotypes and phenotypes but those correlations may not give the expected results; and (3) natural mutations helps to explain the fundamental domains of enzymes and activator proteins, and the characterization of the effects of these mutations on the translated protein have led to a better understanding of normal cell biological processes.

7. Therapeutic efforts When considering a genetic disease, the most important issue is to prevent this disease by pre-natal diagnosis. However, this is not always possible. In this case, the most important issue is the treatment of the disease. Different therapeutic strategies have been developed such as enzyme replacement therapy (ERT), gene therapy, bone marrow transplantation (BMT), chemical chaperones therapy and substrate deprivation therapy. The aim of the therapy is to restore the defective enzymatic activity in the lysosome. This is not so easy for the type of sphingolipidoses with central nervous system involvement. In this situation, the main strategy is to find a therapeutic agent that can penetrate the blood – brain barrier. These strategies have been tested on mouse models and on cultures of patients’ cells and patients [52,53].

Table 2 Genetics and molecular genetics of defective enzymes Defective gene

Structure of the gene

Gene map locus

Number of found mutations

Number of frequent mutations

Inheritance

Ethnic origin

b-Galactosidase Hexosaminidase A Hexosaminidase B GM2 activator a-Galactosidase A Glucocerebrosidase Acid ceramidase Arylsulfatase A Galactosylceramidase Sphingomyelinase

60 kb, 16 exons 35 kb, 14 exons 40 kb, 14 exons 16 kb, 4 exons 12 kb,7 exons 7 kb, 11 exons 30 kb, 14 exons 3.2 kb, 8 exons 56 kb, 17 exons 5 kb, 6 exons

3p21.33 15q23-q24 5q13 5q31.3-33.1 Xq22.1 1q21 8p21.3/22 22q13 14q24.3-32.1 11p15.1-p15.4

29 95 26 4 150

5 3

Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive X-linked recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive

Panethnic Ashkenazi Jewish

8 11 60 60 18

A few A few 3

Ashkenazi Jewish

Panethnic Ashkenazi Jewish

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7.1. ERT Among sphingolipidoses, the non-neuronopathic form of Gaucher disease (type 1) and Fabry disease have been treated by enzyme replacement therapy (ERT) [54]. Placental-derived preparation of the enzyme glucocerebrosidase, aglucerase (Ceredase, Genzyme Inc.), or the recombinant form of glucocerebrosidase, imiglucerase (Cerezyme, Genzyme Therapeutics Inc., Cambridge, MA) are given intravenously to the patients with type 1 Gaucher disease. This therapy has been proven to be safe and effective in more than 3000 patients worldwide [55,56]. The neuronopathic form of this disease could not be treated with ERT because enzyme protein cannot reach the central nervous system by passing through the blood – brain barrier. Recombinantly produced a-galactosidase A in cultured human skin fibroblasts and a-galactosidase A produced by a gene activation technique developed by Transkaryotic Therapies Inc., Cambridge, MA, using a continuous human cell line are used for enzyme therapy in Fabry patients. ERT ameliorates disease symptoms and signs and improves the quality of life [56]. The lifelong intravenous therapy, the costly process and the incapability of passing through the blood –brain barrier are the disadvantages of this therapy [57].

barrier since they are small. Two main classes of inhibitors of GSL biosynthesis have already been described both of which inhibit the ceramide-specific glucosyltransferase. The enzyme catalyzes the transfer of glucose to ceramide, the first step in the biosynthesis of GSLs. The first class of inhibitor is formed by analogues of ceramide. Studies in a knockout mouse model for Fabry disease have shown that oral administration of this compound can result in a marked reduction of the accumulating GSL [55]. The second class of inhibitor is formed by N-alkylated iminosugars. N-alkylated iminosugars, N-butyldeoxygalactonojirimycin (NB-DNJ) and adamantane-pentyl-deoxynojirimycin were used for the treatment of knockout mouse models of Tay-Sachs, Sandhoff and Fabry disease and significant reductions were detected in GSL storage. The first clinical study on the use of NB-DNJ (OGT-918; Oxford Glycosciences) to treat sphingolipidoses was done in 28 type 1 Gaucher patients. Decrease of substrate formation by OGT918 improved the key clinical features of these patients [62]. OGT-918 improved survival of Tay-Sachs and Sandhoff mouse models also [63,64] and this drug enhanced the beneficial effects of BMT [65]. Other inhibitors of glucosylceramide synthase are being developed but they are all in the pre-clinical developmental stage [66 – 68]. 7.4. Gene therapy

7.2. BMT Lysosomal enzymes undergo a cycle of exocytosis and re-uptake by mannose-6-phosphate receptor because of the incompleteness of the binding of lysosomal enzymes to their receptors in the Golgi apparatus. As much as 40% of newly synthesized lysosomal enzymes are secreted depending on the enzyme and cell type. The secreted enzymes can be endocytosed via the plasma-membrane-located receptors by neighbouring cells. In this way, macrophages from bone marrow transplantation (BMT) are able to supply the enzyme to the nervous system by passing through the blood – brain barrier [58,59]. For this reason, this therapy has been used in lysosomal storage diseases for many years [60]. BMT continues to be effective in Gaucher disease, and in mild forms of Krabbe disease, but it has a high morbidity and mortality that limits its use in lysosomal storage disorders [60,61]. 7.3. Substrate deprivation therapy (also termed ‘substrate reduction therapy’) This therapy aims to reduce the rate of GSL biosynthesis. Residual enzyme activity is important for this therapy. Patients who have a significant residual activity could gradually clear lysosomal storage material and therefore should profit most from reduction of substrate biosynthesis. This means that this therapy is suitable for juvenile and adult types of sphingolipidoses. The substances used for substrate deprivation are able to pass through blood – brain

Most of the sphingolipidoses except for Fabry and type 1 Gaucher disease show massive central nervous system involvement. In these diseases, provision of sufficient amounts of enzyme to the nervous system is required. However, a suitable vector is necessary to transfer the normal gene into the central nervous system. Retroviral and adenoviral vectors were developed to transfer a normal gene to the neurons in the cell culture and in the mouse models. Bone marrow stem cells, neural precursor cells or astrocytes are transformed by these viral vectors and given by BMT and direct transplantation into the nervous system, respectively. The injection of viral vectors into the brain directly without any cell transformation is an other possibility. All these possibilities have already been examined in sphingolipidoses [59]. However, the results are not satisfactory and this area requires much more attention before clinical efficacy trials [52]. 7.5. Chemical chaperones therapy Some lysosomal storage diseases appear to be caused by lysosomal enzyme variants that retain catalytic activity but are pre-disposed to misfolding or mistrafficking in the cell. Specific small molecules for defective proteins are able to stabilize the mutant proteins responsible for sphingolipidoses by binding to their native conformation and cause them to pass the quality control system in the ER. More recently, the concept of specifically acting chemical chaperones has been applied to two sphingolipidoses,

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Fabry’s disease and Gaucher’s disease. D -Galactose for Fabry disease and subinhibitory concentrations of inhibitors, (1-deoxygalactonojirimycin for Fabry, N-(n-nonyl)deoxynojirimycin for Gaucher) have been shown to chaperone the enzymes. In the presence of D -galactose in the culture medium of the COS-1 cells and lymphoblasts, a significant increase was observed in the expression of the mutant a-galactosidase A [69,70]. The conformation of the mutant enzyme is stabilized by binding of the D -galactose. An infusion of D -galactose to a patient suffering from the cardiac variant of Fabry’s disease increased the residual enzyme activity from 7 to 10% of normal values over 3 days infusion. After 2 years of treatment, heart transplantation was not required [71]. Studies with subinhibitory concentrations of inhibitors in culture cells of both Fabry and Gaucher disease and animal model of Fabry disease revealed that administration of inhibitors led to a significant increase in the activity of the variant enzymes and to a substantial improvement of therapeutic parameters. These substances can be orally administered, cross the blood – brain barrier and are not so expensive [72 –75].

8. Concluding remarks Over the past decades, biochemical and molecular genetic studies for sphingolipidoses have expanded our understanding of underlying metabolic principles of these diseases and their genes. A new lysosomal digestion model was developed and mechanisms of GSLs hydrolysis within the lysosome were understood. The discovery of the SAPs was an important factor in this process. However, selective degradation of GSLs within the lysosome is still not clear. Additional biochemical and morphological experiments are necessary for the understanding of this process. Various types of disease-causing mutations have been characterized. Among them, a limited number of mutations led to the characterization of active sites, many of them helped to identify the functional domain or post-translational modification sites of the enzymes. Knowledge of the disease-causing mutations is important for treatment of the patients with residual enzyme activity by chemical chaperones in the near future. Although several mechanisms are involved, the pathogenesis of sphingolipidoses has not been fully understood yet. In the near future, sphingolipid research on cell signaling using cDNA microarrays, proteomic and glycomic technologies and animal models will help to investigate the pathophysiology and the development of novel therapies for the treatment of sphingolipidoses. In the past few years, there has been a significant advance in the treatment of type 1 Gaucher and Fabry disease with ERT. In the following years, by increasing clinical trials with different enzyme inhibitors, it may be possible to treat juvenile and adult types of some sphingolipidoses with

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chemical chaperones and substrate deprivation therapy. The identification of a non-toxic long-term vector system for abundant expression of therapeutic proteins with effective delivery to specific regions of the brain remains an immense challenge for gene therapy of the neuronopathic form of sphingolipidoses [76].

Acknowledgements I owe immense gratitude to Prof. Dr Konrad Sandhoff for his kind help and support to my research and for giving me a new perspective in this area.

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