Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA, and 2Department of Medical Genetics, Childrens National Medical Center, Washington, DC 20010, USA
Accepted on August 29, 2000;
Abstract
Glycosphingolipids (GSLs) are plasma membrane components of every eukaryotic cell. They are composed of a hydrophobic ceramide moiety linked to a glycan chain of variable length and structure. Once thought to be relatively inert, GSLs have now been implicated in a variety of biological processes. Recent studies of animals rendered genetically deficient in various classes of GSLs have demonstrated that these molecules are important for embryonic differentiation and development as well as central nervous system function. A family of extremely severe diseases is caused by inherited defects in the lysosomal degradation pathway of GSLs. In many of these disorders GSLs accumulate in cells, particularly neurons, causing neurodegeneration and a shortened life span. No effective treatment exists for most of these diseases and little is understood about the mechanisms of pathogenesis. This review will discuss the development of a new approach to the treatment of GSL storage disorders that targets the major synthesis pathway of GSLs to stem their cellular accumulation.
Key words: glycolipid function/lysosomal storage diseases/neurodegenerative diseases/sphingolipids/substrate deprivation
Introduction
Glycosphingolipids (GSLs) are amphipathic molecules consisting of a ceramide lipid moiety linked to one of hundreds of different externally oriented oligosaccharide structures (Schuette et al., 1999). They can be found in cell-type-specific patterns on the outer leaflet of the plasma membrane of all mammalian cells. GSLs that contain sialic acid residues are known as gangliosides and are most prominent in the nervous system.
GSLs have been implicated in many fundamental cellular processes including growth, differentiation, migration, and morphogenesis. They are thought to contribute to cell-to-cell and cell-to-matrix interactions and in the development and functioning of the nervous system (Hakomori, 1990; Schnaar, 1991
). GSLs may modulate cell signaling by controlling the assembly and interaction of plasma membrane proteins (Hakomori, 1990
; Kasahara et al., 1997
). GSLs may also directly mediate signal transduction through specialized lipid signaling domains (Hakomori et al., 1998
; Iwabuchi et al., 2000
). In another paradigm, GSL metabolites, including ceramide and sphingosine-1-phosphate are believed to be themselves signaling molecules to mediate cellular functions (Huwiler et al., 2000
; Shayman, 2000
).
GSLs are synthesized de novo beginning with the rate-limiting step of ceramide formation at the membranes of the endoplasmic reticulum. On the cytosolic face of the Golgi apparatus UDP-glucose:ceramide glycosyltransferase (GlcCer synthase) transfers a glucose moiety from UDP-glucose to the 1-position of ceramide resulting in the formation glucosylceramide (GlcCer), the committed step in the formation of the majority of GSLs (Figure 1). A less prominent pathway of GSL synthesis is based on the formation of galactosylceramide. Cell-type-specific biosynthesis of more complex GlcCer-based GSLs proceeds on the luminal face of the Golgi with sequential addition of saccharides and transport through the trans-Golgi network to the plasma membrane. Ultimately the GSLs reach the lysosomal compartment by endocytic membrane flow where they are degraded in a step-wise fashion by the concerted action of lysosomal hydrolases and activator proteins. A family of severe diseases is caused by inherited defects in the GSL lysosomal degradation pathway (Figure 2, Table I).
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Targeted disruption of genes in the GSL biosynthetic pathway has yielded new insights into the functional and biological roles of GSLs. Disruption of the gene encoding GlcCer synthase (Ugcg) eliminated the major pathway of GSL synthesis (Yamashita et al., 1999) (Figure 1). Ugcg -/- embryos proceeded into gastrulation with differentiation into the three primitive germ layers. However, embryogenesis was abruptly halted by a major apoptotic process centered within the ectoderm around embryonic day 7.5 during mid-gastrulation. Embryonic stem (ES) cells homozygous for the Ugcg gene disruption and deficient in GlcCer synthase grew normally and were able to form differentiated neural and hematopoietic elements in culture. When these same Ugcg -/- ES cells were injected subcutaneously into mice they were unable to produce mature well differentiated tissues. The results unambiguously demonstrate that GSL synthesis is essential for development and differentiation in vivo, but interestingly not for cell survival, growth, and some aspects of differentiation in vitro. This may reflect a crucial role for GSLs in the complex cellular signaling that operates during embryonic development. The demise of the embryos deficient in GSL synthesis may therefore be the result of absent or inappropriate cellular signals. However, a mechanism whereby the accumulation of toxic sphingolipid metabolites, such as ceramide, directly induces cell death may also be operating.
The functions of gangliosides in the nervous system are only beginning to be elucidated. A direct demonstration of ganglioside function was offered by Sheikh et al. (1999), who used a mouse strain with a targeted disruption of the GalNAcT (ß1,4 N-acetylgalactosaminyltransferase, GM2 synthase) gene and was lacking in complex gangliosides (Figure 1). These mice, which expressed the simple gangliosides GM3 and GD3, displayed decreased central myelination, axonal degeneration in the central and peripheral nervous system, and demyelination in peripheral nerves. These results demonstrate that complex gangliosides play an important role in central myelination and in maintaining the integrity of myelin in the peripheral nervous system. The animals showed a very similar phenotype to mice with a disrupted gene encoding myelin-associated glycoprotein (MAG) (Fruttiger et al., 1995
; Yin et al., 1998
), a receptor that binds complex gangliosides in vitro, suggesting that the underlying defect in the GalNAcT knockout mice involved the elimination of the critical MAG interaction with complex gangliosides.
A similar GalNAcT knockout mouse reported by Takamiya et al. (1996) showed slightly decreased central conduction velocity. The additional finding of male sterility due to azospermia suggested a role for gangliosides in sperm development (Takamiya et al., 1998
). An attenuation of the IL-2 response suggested that complex gangliosides expressed on T cells associate with receptors and other molecules important for IL-2 signal transduction (Zhao et al., 1999
). Even with these significant defects, the knockout mice with a total deficiency of complex gangliosides throughout embryogenesis develop a functioning nervous system, have no gross behavioral disturbances and have a normal life span
Mice with a disrupted galactosylceramide synthase (CGT) gene, can not produce the galactose-based sphingolipids galactosylceramide and sulfatide. These mice are viable but display hindlimb paralysis and tremor (Bosio et al., 1996; Coetzee et al., 1996
; Stoffel and Bosio, 1997
; Dupree et al., 1998
). This severe phenotype is presumably caused by defective myelin sheaths that are thin, incompletely compacted, and unstable with structural abnormalities in the nodal and paranodal regions. The findings indicate that galactosylceramide and sulfatide are essential in myelin formation and maintenance.
Inhibiting GSL synthesis in the treatment of GSL storage disorders
GSL storage disorders are generally caused by inherited errors in lysosomal enzymes or activator proteins that function in the degradation of the GSL substrates. The family of GSL storage disorders includes Tay-Sachs, Sandhoff, Gaucher, and Fabry diseases, GM1 gangliosidosis, Niemann-Pick C, and deficiencies of the activator proteins (Figure 2, Table I). Although individually rare, collectively these disorders are important causes of neurodegeneration in childhood. Each of the disorders has characteristic findings dictated by the nature of the storage material, the cells and tissues affected and the amount of residual degradative capacity (Table I). In many of these diseases, GSLs accumulate to massive levels in cellsparticularly neurons where the synthesis of gangliosides is prominentcausing neurodegeneration and a shortened life span. Other organ systems can also be affected. The mechanism of pathogenesis for these disorders is only beginning to be understood. With the exception of type 1 Gaucher disease, no effective therapy exists for these devastating disorders.
When GSL degradative capacity falls below a critical threshold, the GSL substrate turnover rate is reduced to a level that leads to substrate accumulation and pathologic consequences (Conzelmann and Sandhoff, 1991; Leinekugel et al., 1992
). This critical threshold value is reached when the maximal turnover rate (Vmax) of the degradative system equals the GSL substrate influx rate into lysosomes. In patients cells, residual degradative capacity is produced by mutated but partially active enzyme systems or by alternative degradative pathways. By sufficiently reducing substrate synthesis through the use of inhibitors, the substrate influx into the lysosomal compartment could be balanced with the residual degradative rate leading to a reduction in storage burden and an improvement in clinical outcome. This potential therapeutic approach, termed substrate depletion or substrate deprivation therapy, was first proposed by Radin for the treatment of Gaucher disease (Radin, 1982
).
As a "proof of concept" for substrate deprivation therapy Liu et al. constructed a mouse model with simultaneous defects in synthesis and degradation of GM2 ganglioside (Liu et al., 1999). These doubly mutant mice carried disruptions in both the GalNAcT gene (Figure 1) necessary for the formation of complex gangliosides including GM2 ganglioside, and the Hexb gene encoding ß-hexosaminidase B, the enzyme deficient in Sandhoff disease and responsible for degradation of GM2 ganglioside (Figure 2). These animals showed no complex ganglioside accumulation, vastly improved neurologic function and an increased life span compared with Sandhoff disease mice, validating the concept of substrate deprivation therapy. However, the mice did develop late-onset neurologic disease due to unimpeded accumulation of another class of ß-hexosaminidase substrate, N-linked oligosaccharides. This unmasking of secondary effects following successful elimination of GSL storage further elucidated the pathophysiology of the disorder and predicts that therapeutic efficacy may be incomplete with inhibitors of GSL synthesis in this disorder.
Pharmacologic substrate deprivation
Two classes of GSL synthesis inhibitors have been investigated as possible therapeutic agents for the treatment of GSL storage disorders. Both are competitive inhibitors of the GlcCer synthase. Inhibition at this early common step in GSL synthesis implies that these inhibitors could potentially be effective in treating many of the storage disorders where GlcCer-based GSLs accumulate and contribute to pathogenesis (Figures 1, 2).
The prototype of the first class of GSL inhibitors is the N-alkylated imino sugar N-butyldeoxynojirimycin (NB-DNJ) (Figure 3). NB-DNJ is also a potent inhibitor (micromolar concentrations) of N-linked oligosaccharide processing enzymes -glucosidase I and II and as such NB-DNJ had been found to inhibit HIV replication in vitro (Fleet et al., 1988
; Karpas et al., 1988
). Platt et al. (1994a)
first determined that these compounds are also inhibitors of GlcCer synthase. In vivo, NB-DNJ does not significantly impact glycoprotein processing because a sufficient concentration of the inhibitor is not attained in the lumen of the endoplasmic reticulum, the location of the glucosidases. In contrast, concentrations of NB-DNJ reached in the cytosol are sufficient to inhibit GlcCer synthesis, which takes place on the cytosolic surface of the Golgi apparatus (Platt and Butters, 1998
). The galactose analogue, N-butyldeoxygalactonojirimycin (NB-DGJ) (Figure 3), inhibits GlcCer synthase but shows no inhibition of
-glucosidase I and II in vitro (Platt et al., 1994b
).
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In their initial study Platt et al. demonstrated that the N-butyl and N-hexyl, but not the N-methyl analogs, of NB-DNJ were able to inhibit the biosynthesis of neutral glycolipids and gangliosides (Platt et al., 1994a). The addition of NB-DNJ reduced GM1 expression in all cell lines by 90%. An in vitro model of Gaucher disease was then designed by adding the irreversible inhibitor of ß-glucosidase, conduritol ß-epoxide, to cultured cells resulting in GlcCer accumulation. The addition of 50 µM NB-DNJ to this culture system prevented GlcCer accumulation, suggesting that NB-DNJ might ultimately be useful in the treatment of the group of GSL storage disorders for which derivatives of GlcCer are substrates.
More recently, Shayman and co-workers (Abe et al., 2000a) compared the ability of the pOH-P4 and EtDO-P4 to deplete globotriaosylceramide (Gb3) from Fabry disease lymphocytes. Both compounds at a concentration of 10nM were effective in depleting 7080% of Gb3 from the cells after 3 days of treatment. In contrast, NB-DNJ at 10 µM only minimally lowered Gb3 accumulation over the same time period. At concentrations where the P4 compounds were highly effective in depleting Gb3, cell growth was not inhibited, suggesting that these inhibitors might be potentially useful in treating patients with Fabry disease or related storage disorders.
GSL synthesis inhibitors in vivo
When the use of GSL synthesis inhibitors for the treatment of Gaucher disease was first proposed the long-term effects of chronic GSL depletion on the whole organism were unknown. Following their initial in vitro studies, Platt et al. (1997b) administered NB-DNJ orally at a dose of 2.4 g/kg/day to normal mice for 14 days and found a 5070% reduction in GSL levels in spleen and liver. The large dose was required to achieve sufficient serum concentrations to inhibit GSL synthesis because of poor pharmacokinetics of NB-DNJ in mice (Platt et al., 1997a
). Comparable serum concentration in humans could be reached at a dose of 43 mg/kg/day (Fischl et al., 1994
). Acute toxic symptoms were not observed during long-term administration (118 days) of the drug to mice; however, the mice gained weight more slowly than controls and exhibited a reversible cellular depletion in lymphoid organs (Table II).
The above observations and the findings that total inhibition of complex ganglioside formation by targeted gene disruption had unexpectedly mild consequences strongly suggested that partial GSL depletion using synthesis inhibitors might represent a realistic therapy the GSL storage disorders.
Three recent publications utilizing synthesis inhibitors in mouse models of GSL storage disorders underscore their potential clinical usefulness. The first study utilized the Tay-Sachs disease mouse, a model that displays regional GM2 storage in brain with associated neuropathology but which is relatively asymptomatic (Yamanaka et al., 1994). Oral administration of 4.8 g/kg/day NB-DNJ to the Tay-Sachs model mouse from 4 to 12 weeks of age resulted in a
50% reduction in the storage of GM2 ganglioside in brain and an associated improvement in neuropathology (Platt et al., 1997a
), demonstrating that the inhibitor effectively crosses the blood brain barrier. The efficacy of NB-DNJ was also tested in the Sandhoff disease mouse model (Sango et al., 1995
), which has a more severe phenotype including diffuse GM2 and GA2 storage, progressive neurodegeneration with neuronal apoptosis and a shortened life span. The mice were treated from 3 or 6 weeks of age with 2.4 or 4.8g/kg/day of NB-DNJ, respectively. Treated animals had a delayed onset of neurologic impairment as determined by their ability to perform behavioral tasks, reduced GM2 and GA2 storage in the brain as well as an amelioration of neuronal apoptosis, and an increased life expectancy when compared to age-matched controls (Jeyakumar et al., 1999
). Untreated mice survived until
125 days compared to the treatment group, which survived until
170 days. The side effect of weight loss caused by NB-DNJ treatment (Table II) is a potential issue complicating the life span increase since caloric restriction itself has been shown to lengthen rodent life span (Wanagat et al., 1999
).
Shayman and coworkers (Abe et al., 2000b) administered EtDO-P4 to wild type mice and found a dose-dependent decrease in of GlcCer levels in spleen, liver, and kidney. Kidney GlcCer levels responded very rapidly. A 50% reduction of GlcCer content of kidney was found at an inhibitor dose of 1 mg/kg, given every 12 h for 3 days. A Fabry disease model was used to study the in vivo therapeutic potential of EtDO-P4. This mouse model manifests accumulation of Gb3 in kidney, heart, and liver along with lamellar storage inclusions in kidneys (Ohshima et al., 1997
, 1999). Treatment with 10 mg/kg inhibitor every 12 h for 4 weeks or 8 weeks resulted in significant decreases in renal, cardiac, and hepatic Gb3 accumulation. The drug treatment also caused substantial improvement of the storage pathology of kidneys. After 8 weeks of treatment the mice suffered neither weight loss nor cellular depletion of lymphoid tissues, the described side effects of NB-DNJ (Table II). The effect of EtDO-P4 treatment on GlcCer levels in brain was minimal, suggesting that penetration of this hydrophobic compound across the bloodbrain barrier may be limited.
Clinical trial
Recently, NB-DNJ was administered orally to 28 patients with type 1 non-neuronopathic Gaucher disease who were unable or unwilling to receive enzyme replacement therapy (Cox et al., 2000). Patients received 100 mg of NB-DNJ three times daily, and steady-state plasma concentrations of
1 µg/ml inhibitor were achieved after 46 weeks of treatment. The mean liver and spleen volumes were modestly but significantly lowered by 12% and 19%, respectively, after 12 months, and hematologic parameters improved slightly. Since NB-DNJ causes lymphoid organ acellularity in mice (Table II), it is not known yet how this potential toxicity might influence the reduction of spleen size seen in the patients. The most frequent side effect described in this study was diarrhea in
80% of patients. Two of the patients experienced a peripheral neuropathy that was reversed with discontinuation of the drug (Table II). The clinical improvements in this initial trial are encouraging. Whether or not synthesis inhibitors will be effective in the treatment of GSL disorders with central nervous system (CNS) involvement remains to be investigated.
Potential use of GSL synthesis inhibitors in storage disorders
GM2 gangliosidoses. The GM2 gangliosidoses, Tay-Sachs, Sandhoff, and the GM2 activator deficiency are caused by deficiencies in the or ß subunit of ß-hexosaminidase A or the activator protein, respectively (Gravel et al., 1995
). The infantile, juvenile and adult forms of the disorders differ according to the amount of residual enzyme activity and are determined by the specific genetic mutation. With the exception of hepatosplenomegaly and oligosacchariduria seen in Sandhoff disease, the pathology of the GM2 gangliosidoses is limited to degeneration in the CNS. Infantile forms with the severest enzyme deficiency present in early infancy with visual disturbance and loss of developmental milestones. Death ensues at 24 years of age due to profound CNS degeneration. Onset of the juvenile form in early childhood is associated with a slightly less rapid progression of psychomotor deterioration, spasticity, seizures and death in late childhood. The adult or chronic forms of Tay-Sachs and Sandhoff disease are variable in onset (late childhood to mid-adulthood) and presenting symptoms can include cerebellar ataxia, upper and lower motor neuron disease, peripheral neuropathy and muscle wasting, dystonia, dementia, and psychosis.
Based on the results observed in Tay-Sachs and Sandhoff disease mice treated with NB-DNJ, one might anticipate that GSL synthesis inhibitors represent a potential treatment for the GM2 gangliosidoses with a number of caveats. The drugs would be expected to be more beneficial in adult onset disease where residual enzyme activity is the highest (as much as 10% of normal). Partial GSL synthesis inhibition alone could not eliminate the storage burden in the infantile GM2 disorder since there is very little degradative capacity. Combination therapy using synthesis inhibitors and bone marrow transplantation or some form of enzyme or gene replacement may prove more effective for the infantile disease. In Sandhoff disease the accumulation of oligosaccharides would be expected to contribute secondarily to the clinical phenotype and would not be ameliorated using synthesis inhibitors.
Gaucher disease. Gaucher disease is caused by the deficiency of acid ß-glucosidase required for the conversion of GlcCer to ceramide (Beutler and Grabowski, 1995). Three types have been distinguished. Type 1, the non-neuronopathic form, is characterized by massive hepatosplenomegaly and the presence of GlcCer-storing macrophages (Gaucher cells) in the bone marrow. Additional features include anemia and thrombocytopenia, interstitial lung disease, and skeletal changes (including widening of the marrow spaces particularly of long bones) leading to an increased risk of fracture and deformity. Repeated intravenous infusion of acid ß-glucosidase (alglucerase) enzyme has been very effective at decreasing the storage burden in liver and spleen, correcting the hematologic abnormalities, and arresting the progressive skeletal deformities. It is unclear whether enzyme therapy can reverse the skeletal changes or prevent the lung disease found in a minority of patients.
Type 2 Gaucher disease, the acute neuronopathic form, is characterized by progressive CNS involvement manifesting in the first few months of life. Failure to thrive, laryngospasm, hepatosplenomegaly, and recurrent infection are also common. Progression is rapid resulting in spasticity, fixed strabismus, and retroflexion of the neck with limb rigidity. Seizures are common with death occurring by age 2. More severe phenotypes manifesting as hydrops fetalis or severe lamellar icthyosis have also been described previously (Stone and Sidransky, 1999). Enzyme replacement with alglucerase has not been successful in reversing the neurologic progression even when initiated in the newborn period. (Prows et al., 1997
).
Type 3 Gaucher disease, the subacute neuronopathic form, is intermediate in severity between types 1 and 2. Hepatosplenomegaly and hematologic manifestations usually appear within the first 2 years of life, with neurologic findings often including an eye movement disorder developing in the first decade. Death usually occurs in the second decade. Alglucerase therapy has been effective at reducing the storage burden in type 3 patients although higher doses of enzyme are required. Reversal or stabilization of the progressive neurologic involvement with enzyme therapy has not been well documented. The lack of efficacy in type 2 patients suggests that alglucerase does not adequately cross the blood brain barrier to provide therapeutic efficacy.
Modest efficacy has been achieved using NB-DNJ in a small clinical trial with type 1 Gaucher patients as discussed above (Cox et al., 2000). The combination of enzyme replacement and synthesis inhibitor therapy may have additional advantages. Enzyme replacement would be expected to initially lower the storage burden and synthesis inhibitors might subsequently prevent substrate accumulation. The result could be a lowering of the required enzyme dose and/or a decrease in the frequency of administration, thereby lowering the cost and improving patient compliance. Assuming penetration across the bloodbrain barrier, the addition of a synthesis inhibitor to the therapeutic regimen in patients with type 3 disease may lower the storage burden in the CNS, thereby preventing the eye movement disorder and developmental delays seen in these patients.
Fabry disease. In contrast to many GSL storage disorders, Fabry disease does not feature CNS dysfunction (Desnick et al., 1995). Fabry disease is X-linked recessive implying a 50% risk for male offspring of hemizygous carrier females. The deficient enzyme is
-galactosidase A. GSLs, predominantly Gb3, with terminal
-galactosyl groups accumulate as lamellar inclusions in blood vessels, smooth muscle, kidney, cornea, and peripheral nerves. The pathologic changes are reflected in the clinical symptoms which include peripheral neuropathy with painful paresthesias of the hands and feet, mitral valve prolapse, aortic stenosis and thickening of the left ventricle and intraventricular septum, and small vessel disease causing cerebral thrombosis, myocardial infarction, visual impairment, and renal failure. Characteristic of the disorder are angiokeratomacutaneous blood-filled papulesfound over the penis, scrotum, and umbilical areas. Clinical trials using replacement enzyme are currently underway in a limited number of patients. The initial success of short-term administration of EtDO-P4 in the Fabry mouse model suggests that GSL synthesis inhibitors alone or in combination with enzyme replacement may also be beneficial in Fabry patients. The pharmacokenetic profile of these inhibitors suggest that the kidney, a site of major pathology in Fabry disease, may be a preferential target.
GM1 gangliosidosis. GM1 gangliosidosis is a less frequent but clinically severe error in the degradation of complex gangliosides and is caused by decreased activity of lysosomal enzyme GM1 ß-galactosidase (Suzuki et al., 1995). Severe infantile and adult forms are described, as well as a juvenile form (type 2) limited to brain involvement. In contrast to the infantile GM2 gangliosidosis, the infantile GM1 gangliosidosis patients appear abnormal at birth with non-pitting edema of the face and extremities and facial dysmorphism. A cherry red macula is present in 50% of patients. Progressive systemic involvement includes hepatomegaly, modest splenomegaly, and skeletal features similar to dysostosis multiplex seen in patients with mucopolysaccharidosis. Seizures beginning within the first 6 months of life dominate the clinical presentation. Death generally occurs by 2 years of age. The juvenile form of GM1 gangliosidosis has a more prolonged course and the adult type is slowly progressive and characterized by the extrapyramidal signs of rigidity and dystonia. Adult patients have only minimal visceral involvement. GSL inhibitors might be expected to decrease GM1 and GA1 storage in the brain, but would not be expected to impact the storage of keratan sulfate or oligosaccharides producing the skeletal changes or visceromegaly respectively. One might predict that enzyme replacement therapy with ß-galactosidase would improve the visceral and skeletal manifestations. In adult patients with residual enzyme activity, a combination of enzyme replacement and synthesis inhibitors might be efficacious.
Niemann-Pick disease Type C. Niemann-Pick disease Type C (NP-C) disease is an inherited lipid storage disorder affecting both the visceral organs and the nervous system (Pentchev et al., 1995). The disease is caused by mutations at either the NPC1 or the NPC2 locus. Unlike other lipid storage diseases, which are caused by mutations in genes encoding enzymes and activator proteins required for lysosomal degradation of lipid substrates, NP-C disease is caused by an error in intracellular trafficking. The cellular defect in NP-C disease impairs the redistribution of exogenously derived cholesterol, leading to its accumulation in lysosomes and in the trans-Golgi apparatus. However, the cellular accumulation of a variety of endogenously synthesized lipids including GSLs occurs in NP-C tissues. In the CNS, a site of major pathology, GSLs, both neutral glycolipids and gangliosides, are the predominant storage material. The prominent neuronal ganglioside storage in NPC disease has been implicated as a possible factor causing CNS dysfunction (Siegel and Walkley, 1994
; Walkley et al., 1995
, 1998).
The age of onset in NP-C varies from perinatal to adulthood with the late infantile and juvenile forms constituting 6080% of patients. Vertical supranuclear gaze palsy is a hallmark of NP-C patients who also present with hepatosplenomegaly, ataxia, dysarthria, dystonia, seizures, and progressive dementia. Adult onset NP-C, responsible for 5% of cases, has a much more variable clinical presentation including psychiatric symptoms. Like other GSL storage disorders, an earlier age of onset correlates with a more rapid neurodegenerative decline and shortened life span. (Vanier and Suzuki, 1998).
To determine if complex ganglioside storage is a pathogenic factor in NP-C disease, NP-C mice were established with targeted mutations in the GalNAcT gene (Liu et al., 2000). By virtue of the GalNAcT mutation these double mutant animals did not accumulate the GM2 ganglioside or GA1 and GA2 glycolipid found in the CNS of the NP-C mice. Although much of the neuronal storage pathology was relieved, there was no improvement in clinical phenotype or life span demonstrating that complex ganglioside accumulation is not primarily responsible for the neurodegeneration observed in NP-C disease.
Abnormal storage lipids that remained in the double mutant mice included GM3, lactosylceramide and GlcCer, raising the possibility that the smaller GSLs may contribute to the neuropathogenesis in NP-C disease If so, treatment with inhibitors of GlcCer synthase could ultimately prove to be useful for therapy.
Sphingolipid activator protein deficiencies. Sphingolipid activator protein (SAP) deficiencies are very rare disorders resulting in GSL accumulation (Sandhoff et al., 1995). SAPs are small nonenzymatic glycoproteins necessary for the hydrolysis of a variety of glycolipids by specific acid hydrolases. Two SAP genes have been described. The first, GM2 activator, produces a protein that forms complexes with, and is essential for the hydrolysis of GM2 by ß-hexosaminidase A. The GM2 activator deficiency is indistinguishable from infantile Tay-Sachs disease in clinical phenotype. GSL synthesis inhibitors would be expected to be equally effective in both diseases.
The other four SAP proteins derive from a single common precursor protein that is processed into SAP-A, B, C, and D within lysosomes. SAP-B is a nonspecific glycolipid binding protein that stimulates hydrolysis of glycolipids, most notably sulfatide by arylsulfatase A.
SAP precursor deficiency has been described (Table I). This disease was extremely severe with death at 16 weeks after birth and with abnormalities observed in the fetal stage (Harzer et al., 1989) making this syndrome an unlikely candidate for therapy.
Mutations in SAP-B result in the accumulation of sulfatide and other sphingolipids. The clinical phenotype is similar to juvenile metachromatic leukodystrophy and includes seizures, spasticity, and progressive loss of motor and cognitive milestones. In affected patients macrophages are filled with metachromatic material indicative of sulfatide storage while neuronal storage is non-metachromatic but shows periodic acid-Schiff-positive membranous bodies of the gangliosidosis type. Synthesis inhibitors might retard the neuronal GSL storage and improve CNS function, however, the storage of sulfatide would likely be unaffected.
SAP-C is essential for the degradation of GlcCer and its deficiency results in the accumulation of GlcCer in the reticuloendothelial system and a disorder similar to Gaucher type 3 with juvenile visceromegaly, skeletal manifestations and neuronopathic disease. As with Gaucher disease, GSL synthesis inhibitors may improve the clinical phenotype by slowing the accumulation of GlcCer.
Conclusions
Significant progress has been made in the use of synthesis inhibitors as therapy for the GSL lysosomal storage diseases. Beginning with the initial concept by Radin, the research was spurred on by the identification of small molecule inhibitors and the establishment of suitable animal models for testing and has culminated in the first clinical trials in Type 1 Gaucher patients. This clinical study showed a positive, albeit modest, clinical improvement in the small cohort of patients. The study also showed that the treatment could be tolerated for at least the short term. These results warrant investigating the use of inhibitors in other GSL storage disorders, including those with CNS involvement.
Based on the research thus far some principles have emerged regarding this new approach to therapy. As implied in the threshold model of lysosomal storage activity, balancing the rates of substrate influx and degradation is essential. This would be far easier to attain with inhibitor therapy in juvenile and adult patients, who have significant levels of residual activity than in infantile patients who have little. In the case of the infantile diseases, augmentation of enzyme levels by gene, enzyme, or cell replacement would be expected to enhance the effectiveness of inhibitor therapy. Many of the GSL disorders also involve the storage of non-GSL substrates. GSL synthesis inhibitor therapy would not be expected to alleviate this storage and its use clinically may unmask additional symptomatology. Ultimately the effectiveness of the inhibitor therapy for many of severe disorders which have CNS involvement will depend on the ability of the drug to cross the bloodbrain barrier and lower neuronal substrate levels, an issue not yet fully resolved.
An important question that remains unanswered is the consequences of long-term GSL synthesis inhibition. While it is clear that total synthesis inhibition is not desirable as illustrated by the knockout mice in GSL synthesis pathways, it is not known at what level synthesis inhibition can be tolerated without impacting the normal functions carried out by GSLs. This final issue cannot be fully addressed until the normal functions of GSLs are more completely understood.
Acknowledgments
We thank Donna Krasnewich and members of the Proia lab for comments on the manuscript and April Howard for creating the figures.
Abbreviations
Glycosphingolipid nomenclature is that of Svennerholm (1994); GSLs, glycosphingolipids; CNS, central nervous system; GlcCer synthase, UDP-glucose:ceramide glycosyltransferase; GlcCer, glucosylceramide; ES cells, embryonic stem cells; GalNacT, ß1,4 N-acetylgalactosaminyltransferase; MAG, myelin-associated glycoprotein; NB-DNJ, N-butyldeoxynojirimycin; NB-DGJ, N-butyldeoxygalactonojirimycin; PDMP, (R,R)-(D-threo)-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; P4, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; EtDO-P4, D-threo-1-(3',4'-ethylenedioxy) phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; pOH-P4, D-threo-4'-hydroxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; Gb3, globotriaosyl-ceramide; NP-C, Niemann-Pick disease Type C; SAP, sphingolipid activator protein.
Footnotes
1 To whom correspondence should be addressed at: National Institutes of Health, Building 10, Room 9N314, Bethesda, MD 20892. Copyright does not extend to this article, which is a work of the United States government.
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