Genetic Studies in Model Organisms
GSLs are essential for the development of multicellular organisms. Mutation and/or silencing of the gene encoding the GlcT enzyme, a unique enzyme that regulates the production of glucosylceramide, is lethal in C. elegans, D. melanogaster, and M. musculus (Yamashita et al., 1999; Marza et al., 2009; Kohyama-Koganeya et al., 2004) (Tables 1 and 2; Fig. 2). In particular, mouse embryos undergo a massive apoptosis following their gastrulation, possibly due to the accumulation of ceramide that is predicted to occur in the absence of glucosyltransferase (Hannun and Obeid, 2008). Along this idea, accumulation of ceramide molecules upon mutation of the neutral ceramidase gene leads to the degeneration of Drosophila photoreceptors (Acharya et al., 2008).
Table 1. List of the Different Genetically Engineered Mice for GSL Synthases
|Affected GSL product||Enzyme name||Phenotype||Reference|
|Glucosylceramide (GlcCer)||Glucosylceramide synthase||Following gastrulation, embryos die and exhibit a massive apoptosis.||Yamashita et al. (1999)|
|Lactosylceramide (LacCer)||β4Gal-T6 or β4Gal-T5||Embryos die early (E10), most likely because of a defect in the development of extra- embryonic tissue. The mutation also affects O- and N-glycoprotein synthesis.||Kumagai et al. (2010); Nishie et al. (2010)|
|GM3||GM3 synthase (Siat9 enzyme) Sialyl transferase||Mice exhibit hearing loss and a hypersensitization of the insulin pathway, which confer to them a resistance to an induced high-fat diet and the development of a subsequent diabetic phenotype. Mice survive more than 1 year.||Yamashita et al. (2003); Yoshikawa et al. (2009)|
|Gb3 Globoside||Gb3 synthase||Mice seem to develop normally and have, in addition, a resistance to invasion by the Shiga toxin.||Okuda et al. (2006)|
|GA2 or GM2 or GD2||N-acetyl-galactosyl transferase or GM2 synthase (Galgt1)||Mice are viable and survive beyond 1 year of age, displaying slow axonal degeneration and locomotor deficits. Males are sterile.||Takamiya et al. (1996); Sheikh et al. (1999); Liu et al. (1999)|
|GalCer||Galactosylceramide synthase||Mice develop almost normally but quickly start to show hind limb weakness, ataxia, and neurodegeneration. Males are sterile, most likely because of the lack of specific testis lipids, the seminolipids.||Coetzee et al. (1996); Honke et al. (2002)|
|GlcNAcGal-Glc- ceramide||ß3GnT-5||Mice are viable and fertile; 80% of them die between 5–15 months with splenomegaly, enlarged lymph nodes, fur loss, and obesity phenotypes. B cell function is also affected.||Togayachi et al. (2010)|
|GD3||GD3 synthase||Mice are viable and fertile.||Okada et al. (2002)|
|GD3/GM2/GD2||Double KO combination||Mice develop and live without a major problem until they suddenly die around 12 weeks of age with skin and epithelium lesions as well as CNS and PNS degeneration. It has been proposed that ganglioside deficiency may activate ectopic complement activation that would favor the induction of neurodegeneration.||Inoue et al. (2002); Ohmi et al. (2009)|
|GM3/GM2/GD2||Double KO combination||Mice develop according to Mendelian frequency but start to develop ataxia, hind limb weakness, and uncoordination, which increase with age. A minimal percentage of mice survive beyond 5 months. Mice have been shown to display a high level of lactosylceramide and lactosylceramide-3- sulfate, which have been correlated with catastrophic neurodegeneration associated to vacuolar cytosol observed especially in oligodendrocytes. The phenotype is reminiscent of GalCer synthase mutation.||Yamashita et al. (2005)|
Table 2. Phenotypic Characteristics of GSL Synthase Mutants in Invertebrates
|Species||Glycosphingolipid product||Enzyme name||Phenotype||Reference|
|D. melanogaster||Glucosyl ceramide||GlcT||Embryos die, exhibiting a massive apoptosis upon gene silencing (upon dsRNA injection).||Kohyama-Koganeya et al. (2004)|
|Mactosylceramide/ Man-Glc-Cer (MacCer)||Egh||Zygotic mutants develop until late larval/pupal stages and exhibit perturbation in photoreceptor axogenesis. Maternal mutants are mildly neurogenic and lack their ventral ectoderm. Hypomorphic mutations allowing adult viability are associated with perturbation of the courtship behavior.||Ellis and Carney (2011); Fan et al. (2005); Goode et al. (1996a); Rubsam et al. (1998); Soller et al. (2006); Wandalll et al. (2003, 2005).|
|GlcNAcβ1-3Manβ1- 4Glcβ1-Cer (Ap3Cer)||Brn||Zygotic mutants develop until late larval/pupal stages. The development of follicular tissue is affected and this phenotype has been associated with perturbations in the gradient formation of one of the EGFR ligands in Drosohila as well as Notch ligand loss of function. Maternal embryonic mutants exhibit a mild hyperplasia of the nervous system and a hypoplasia of their epiderm. Brnfs107 hypomorphic alleles have a locomotion defect similar to β4GalNAcTA mutants (see below).||Chen et al. (2007); Goode et al. (1992, 1996a, 1996b); Wandall et al. (2005); Schwientek et al. (2002); Pizette et al. (2009)|
|Complex GSLs||β4GalNAcTA β4GalNAc TB||Double mutants are viable. β4GalNAcTA defective flies are uncoordinated and exhibit locomotion defects, most likely associated with impairment of their neuromuscular junction formation. β4GalNAcTB loss of function affects specifically follicular epithelium morphogenesis leading to the fusion of embryonic dorsal appendages.||Chen et al. (2007); Haines and Irvine (2005); Johswich et al. (2009); Stolz et al. (2008).|
|Complex GSLs||α4GalNAcT1/ α4GalNAcT2||Double KO are viable and fertile.||Hamel et al. (2010)|
|C. elegans||Glucosylceramide||GlcT||Mutation is lethal and rescued by expressing the protein in a subset of intestinal cells.||Marza et al. (2009)|
|Mactosylceramide- MacCer||Bre 3||No developmental defect but mutation rescues a Notch gain of function.||Griffitts et al. (2005)|
|GlcNAcβ1-3Manβ1- 4Glcβ1-Cer- Cer or Ap3Cer||Bre 5||No developmental defect but mutation rescues a Notch gain of function.||Katic et al. (2005); Griffitts et al. (2005)|
|Complex GSL||Bre 4||No developmental defect noticed.||Griffitts et al. (2005)|
Genetic analysis of subsequent enzymes in the GSL synthesis pathway has provided interesting insights about the in vivo role of GSLs. Deletion of the genes involved in the synthesis of MacCer in invertebrates (corresponding functionally to LacCer in vertebrates) (Wandall et al., 2005) is compatible with an apparent normal life in C. elegans (Katic et al., 2005) but not in Drosophila (Goode et al., 1992, 1996b) (Table 2). In mice (Fig. 2 and Table 1), genetic redundancy and functional diversification may complicate the issue. Indeed, two enzymes appear to regulate the production of LacCer. Additionally, these enzymes are also involved in both O- and N-protein glycosylation (Yamashita et al., 1999). Hence, it is difficult to evaluate the specific impact of LacCer deletion in vertebrate membranes. Simple model organisms, therefore, constitute a precious source of information concerning the role of MacCer and LacCer molecules in development. In Drosophila, MacCer is synthesized by a glycosyltransferase known as Egghead (Egh) (Fig. 2 and Table 2; Wandall et al., 2003). While mutations in the egh gene result in the accumulation of GlcCer lipids, mutations in the gene encoding the enzyme responsible for the glycosylation of MacCer into GlcNAcMacCer, known as Brainiac (Brn) in flies (Fig. 2 and Table 2), lead to the accumulation of both MacCer and GlcCer lipids (Wandall et al., 2005; Pizette et al., 2009). Interestingly, egh and brn mutants exhibit very similar phenotypes that may result from either the accumulation of GlcCer lipids (or any product upstream of GlcCer in the sphingolipid synthesis pathway) or the depletion of complex GSLs (i.e., any product downstream of MacCer in the GSL synthesis pathway). While individuals carrying strong egh and brn alleles die at late pupal stages with no obvious anatomical perturbations, hypomorphic mutant flies are viable but female sterile. Genetic mosaic analyses have revealed that the synthesis of complex GSLs is required in the female germ-line for proper oogenesis (Goode et al., 1992, 1996a, b). In particular, egh and brn mutant oocytes fail to induce the correct proliferation and epithelial morphogenesis of the wild-type somatic follicular cells that surround the oocyte, resulting in defects similar to those seen upon decreased Notch and/or EGFR signaling (Goode et al.1992, 1996a, b; Pizette et al., 2009). Interestingly, the worm homologues of both egh and brn also modulate cell–cell communication mediated by Notch (Katic et al., 2005). We discuss below how egh and brn mutations might affect such essential signaling networks at the molecular level.
LacCer molecules can be further modified in mice by four different enzymes (Fig. 2 and Table 1) to generate, either GM3 (the precursor of all gangliosides), Gb3 (the first GSL of the globoserie), GA2 (the precursor of all asialo-serie GSL), or GSLs from the lacto/neolacto-serie. Interestingly, these four enzymes are developmentally regulated (Xu et al., 2010). Of note, the product of Brn may be either considered as a neutral GSL similar to Gb3 or as an acidic GSL similar to GM3 when covalently linked to phospho-ethanolamine (PE) molecules (Chen et al., 2007). While brn is an essential gene in flies (but not in worms), none of the four enzymes is strictly required for mouse embryonic development, at least individually (Table 1) (Togayachi et al.2010; Takamiya et al., 1996; Sheikh et al., 1999; Yamashita et al., 2003; Okuda et al., 2006; Yoshikawa et al., 2009). This may suggest that the GM3/GA2, Gb3, and Lacto- GSLs are to a certain extent interchangeable during the developmental period as well as after birth (Tables 1 and 2). Mutant mice, however, exhibit specific deleterious phenotypes, suggesting that compensation between the distinct GSL series is not complete (Table 1). Of interest, the GM3 synthase mutant mice exhibit a specific degeneration of the Conti organ and are believed to become deaf early after birth (Yoshikawa et al., 2009).
Finally, we note that the loss of a GSL species may result in the increase of other GSL species from alternate branches (Yamashita et al., 2005; Furukawa et al., 2008) More generally, lipidomic studies in yeast and flies have indicated that compensatory changes maintain cellular homeostasis upon loss of specific lipid species (Guan et al., 2009; Carvalho et al., 2010). Thus, deducing the role of a given GSL species only from loss-of-function studies may be misleading and, obviously, genetic data are best interpreted in the context of complementary molecular data. Also, future lipidomic studies of GSL mutants assessing expression of the various enzymes involved in sphingolipid/GSL metabolism and the relative amount of their molecular products should provide important insights into in vivo sphingolipid homeostasis.
GSLs in Neurological Diseases
This impact of GM3 loss on mouse hearing illustrates the importance of gangliosides in nervous system development. In flies, egh and brn mutant embryos display a mild hyperplasia of the central nervous system and of the chordotonal neurons in the peripheral nervous system (Goode et al., 1996a; S.P. and F.S., unpublished results). Interestingly, chordotonal organs in flies and Conti organ neurons in mammals are specified by a conserved proneural transcription factor called Atonal in flies and Math1 in mice (Ben-Arie et al., 2000; Wang et al., 2002). The lineage specificity seen in flies may constitute an important advantage to determine the identity of the GSL-sensitive factors in Atonal/Math-derived neurons in mammals.
Neurodegenerative phenotypes reflecting the essential role of complex GSLs and gangliosides in the maintenance of neuron homeostasis have been observed in various GSL mutant genetic backgrounds. Mice deleted either for GM2 and Gd3 or GM2 and GM3 synthases develop normally but start to display hind limb weakness, ataxia, and a global neurodegeneration between 3 and 12 weeks of age (Inoue et al., 2002; Yamashita et al., 2005; Ohmi et al., 2009). Knocked-out mice for both GM3 and GM2 synthases do not form gangliosides and display the strongest phenotype. In the majority of the cases, the mice die around 2 months of age with unusually high levels of lactosylceramide and lactosylceramide sulfate (Yamashita et al., 2005). Of interest, the accumulation of MacCer products observed in hypomorphic brn flies (Pizette et al., 2009, Wandall et al., 2005) is accompanied by locomotion defects (Chen et al., 2007). Similarly, mutant flies for the β4GalNAcTA enzyme that catalyzes the production of more complex GSLs (Fig. 2) also present locomotion defects and abnormal development of their neuromuscular junctions (Haines and Irvine, 2005; Chen et al., 2007; Haines and Stewart, 2007; Table 2). Whether or not these various mutants display neurodegenerative features will be important to establish.
In any case, these studies illustrate how animal models may further our understanding of neurodegenerative diseases observed in GSL Storage Disorders as well as in a number of neurodegenerative diseases, such as Parkinson or Alzheimer diseases during which GSL metabolism is also perturbed. The role of GSLs in these pathologies has already been reviewed (Ginzburg et al., 2004; Xu et al., 2010) and we refer the reader to these reviews for a detailed analysis. Here, we simply wish to emphasize that GSL genetic models may constitute powerful tools in order to (1) clarify whether cellular degeneration may be hampered by reducing GSL accumulation; (2) define putative genetic and/or pharmacological targets allowing to compensate for such accumulation and help neurons to reacquire their normal homeostasis (Ginzburg et al., 2004); and (3) set regenerative strategies. This late idea is exemplified by the improved regeneration of hearing neurons in deaf cats observed following GM1 adjunction (Leake et al., 2007), which is believed to increase the autocrine expression of neurotrophic factors and lead to TRK receptor stimulation (Rabin et al., 2002; Mallei et al., 2004).