Mice Expressing Only Monosialoganglioside GM3 Exhibit Lethal Audiogenic Seizures

Abstract

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 ␤1,4-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.Gangliosides are glycosphingolipids that contain sialic acid (reviewed in Ref. 1). They are found on the external leaflet of the plasma membrane on eucaryotic cells and are most abundant in the central nervous system (CNS) 1 where they represent the major sialoglycoconjugate. Because of their dramatic changes in expression during neuronal development and differentiation (3-6), as well as their prominence in the mature CNS, gangliosides have long been assumed to have fundamental roles in CNS development and function.In the ganglioside biosynthetic pathway (1) (see Fig. 2A), lactosylceramide serves as the core structure. The first ganglioside synthesized, GM3, 2 is produced by the transfer of an ␣2,3-linked sialic acid residue to lactosylceramide. Subsequently, GM3 can be modified by the action of ␤1,4-N-acetylgalactosaminyltransferase (GalNAcT, EC 2.4.1.92) to produce GM2 and other complex gangliosides. Alternatively, GM3 can be modified by the action of GD3 synthase (CMP-sialic acid: GM3 ␣-2,8-sialyltransferase, EC 2.4.99.8) to produce the disialoganglioside GD3, which diverts the pathway to the synthesis of b-and c-series gangliosides. Gene targeting in mice has been a particularly fertile approach for uncovering the functions of gangliosides in the CNS. Disruption of the GalNAcT gene (7) blocks the synthesis of complex gangliosides and results in the expression of only the simple gangliosides GM3 and GD3. Surprisingly, these mutant mice are viable, with a normal life span and a CNS that is largely intact both morphologically and functionally (8, 9). These mice do, however, exhibit an agerelated dysmyelination process that is associated with axonal degeneration (10). The mechanism for dysmyelination may be the absence of neuronal ganglioside ligands for myelin-associated glycoprotein (MAG) resulting in myelin instability. Ultimately, motor defects are observed in aged, 12-month-old GalNAcTϪ/Ϫ mice, suggesting a role for complex gangliosides in long-term CNS maintenance ...

Sphingolipids and membrane biology as determined from genetic models

“…The transfer of sialic acid to lactosyl ceramide forms GM3, which serves as substrate for two different pathways. The product of GD3 synthase action on GM3 is GD3 and that of β1,4-N-acetylgalactosaminyltransferase (also known as GalNAcT EC 2.4.1.92) is GM2 and other complex gangliosides [125]. Table 1 lists some of the knock-out mouse lines for sphingolipid-related genes that have been generated over the last two decades.…”

“…Defective sphingolipid metabolism can also affect neuronal function directly. It is interesting to note that while sialyltransferase (GD3 synthase) knockout mice are homozygous viable without clinical defects, a double knockout of β1,4-N-acetylgalactosaminyltransferase and GD3 synthase results in sudden death and susceptibility to seizures, indicating altered membrane properties [125]. GM3 synthase knock-out mice have a hypersensitive insulin response, and progress to hypoglycemia faster than wild-type mice when challenged with parenteral insulin.…”

Gangliosides help stabilize the brain

“…Adding to the complexity of potential mechanisms is the severe neurodegeneration in mice with mutations in both Siat9 and Galgt, which are totally deficient in the common brain gangliosides (R.L.P., unpublished data). Though long invoked as a possible function of gangliosides, brain development seems to be largely normal in ganglioside-deficient mice 7,10,[12][13][14] , suggesting that developmental defects are unlikely to be the underlying cause of the disorder, though subtle defects have yet to be ruled out.…”

“…GM3 Synthase (Siat9) Mice with mutations in both Galgt1 and Siat8a have a block after GM3 ganglioside synthesis 10,15 . Mice with mutations in both Siat9 and Galgt1 have a block after lactosylceramide (LacCer) synthesis (R.L.P, unpublished data).…”

“…Mice carrying mutations in two ganglioside glycosyltransferase genes, Galgt1 and Siat8a, which truncate the synthesis pathway after the production of GM3 ganglioside, are markedly susceptible to sound-induced tonic-clonic seizures 10 . This leaves us with the apparently confounding situation in which individuals that do not synthesize any GM3 ganglioside and mice that synthesize only GM3 ganglioside both suffer seizures.…”